b> CAREL PETRUS JOOSTE S u b m itted in f u l f il lm e n t of the re

Transcription

ISOLATION AND LIMITED CHARACTERI SAT 1ON OF
LION - P a n t h e r *
Uo
- ALBUMIN
b>
CAREL PETRUS JOOSTE
S u b m itte d
for
in f u l f i l l m e n t o f
th e r e q u ir e m e n ts
t h e D e g re e o f M a s t e r
of S c ie n se
i n th e
D ep artm en t of G eneral
S cho ol
P h y sio lo g y
o f Dei t i s t r y
U n i v e r s i t y o f th e W i t w a t e r s r a n d
1987
Jo hannesburg
1987
ISOLATION AND LIMITED CHARACTERISATION OF
LION - P a n t h e r s
t e o - ALBUMIN
by
CAREL PETRUS JOOSTE
S u b m itte d
fo r
in f u l f i l l m e n t o f t h e r e q u i r e m e n t s
t h e D e g re e o f M a s t e r o f S c i e n c e
in th e
D ep artm en t of G en e ra l
P h y sio lo g y
Scnool of D e n t i s t r y
U n i v e r s i t y of th e W itw a te rs ra n d
1987
Johannescurg
1987
PREFACE
The e x p e r i m e n t a l
th e D e p a rtm e n t
work d e s c r i b e d
o f G eneral
in t h i s d i s s e r t a l o n w as c o n d u c t e d
P h y sio lo g y ,
U n iv e r s ity of W itw a te rs ra n d
under
th e s u p e r v i s i o n of P r o f e s s o r Jo h a n n H a t t i n g h .
These
stu d ie s
su b m itte d
r e p r e s e n t o r i g i n a l work by t h e a u t h o r and h a v e n o t been
in a n y fo rm t o a n o t h e r U n i v e r s i t y .
t h e w o rk o f o t h e r s
C .P .
Where u s e w as made of
i t h a s b e e n d u l y a c k n o w le d g c c .
JOOSTE
L i™
in
day of
, 19 S t
in
th e t e x t .
ill
ABSTRACT
On routine plasm a p ro tein analysis of a v ariety of wild animal species, the
ele c tro p h o re tic s e p a r a tio n p a tt e r n of lion albumin was found to be peculiar in
th e sen se t h a t it c o n siste d of a double band of unequal concentration. This
pnenomenon w as in v e s tig a te d in the following way:
1.
Albumin w as is o la te d from lion plasma by two d if fe re n t methods.
2.
A v arie ty of d if fe re n t analytical methods were employed to d em onstrate the
albumin p a t t e r n . In th is reg ard cellulose ac e ta te and disc gel elec tro p h o resis
w ith and w ithout SDS as well as analytical and p re p a ra tiv e iso electric focusing
and column exclusion chromatography were used,
3. Combinations of th es e analytical methods were used to ascertain whether the
p a rtic u la r s e p a r a ti o n p a t t e r n was influenced by:
a) geographic origin of donor lions,
b) time period betw een lion immobilization and sample taking,
c) various immobilizing drugs,
d) specific anticoagulant,
e) sample sto rin g procedures,
f) length of elec tro p h o retic sep aratio n time,
g) n u tr itio n a l s t a t u s of donor lions,
h) degree of concentration of sample,
i) wild or captive s t a t e of donor lions.
/l.
Specific a n tib od ies to lion albumin were ra ise d for immunological
c h a r a c t e r i s a ti o n of th is p ro tein .
I t w as concluded th at:
i) the se p a r a tio n p a tt e rn w as indeed due to albumin,
ii) the elec tro p h o re tic picture was not influenced by analytical method or by
sample handling,
iii) the two albumin bands exhibit d ifferen ces in charge but not in molecular
m ass,
iv) the two bands are immunologically similar.
ACKNOWLEDGEMENTS
END OF THIS STUDY MY SINCERE THANKS AND DEEPEST APPRECIATION GO TO
• Johann H a t t i n g h f o r having the utmost p a t i e n c e f or t h i s
p a r t - p r a c t i t i o n e r p a r t - t e a c h e r w it h academic a m b i t i o n .
. E l a i n e Marcus f o r a l w a y s , from c a pt u r e t o c o n c l u s i o n ,
being t h e r e t o h e l p .
• Tracy Marcus f o r spending h o l i d a y hours t o type b o r ing
chapters.
. The r e s e a r c h s t a f f o f:
Kruger N a t i o n a l Park
Et osha Game Reserve
J ohannesburg Zoo
f o r h e l p i n g with sample c o l l e c t i n g .
. The s t a f f of the Department ot Ge ne ra l P h y s i o l o g v f o r
c r e a t i n g a p l e a s a n t environment t o work i n .
CONTENTS
INTRODUCTION.
A.
SOLUBILITY
SOLUTION.
1.
Mol e c u ’ a r s i z e
o-f t h e p r o t e i n .
2.
Amino a c i d c o m p o s i t i o n ,
s e q u e n c e and co n -form a t i on
o-f t h e p r o t e i n .
3.
C harge of
th e p r o t e i n .
4.
S o lv en t d i e l e c t r i c
5.
Io nic
6.
T em p eratu re .
c o n sta n t.
stre n g th .
B.
EXPERIMENTAL MANIPULATION OF PROTEINS.
C.
APPROACH OF THE PRESENT STUDY.
MATERIALS AND METHODS.
A.
B.
ISOLATION OF ALBUMIN FROM LION WHOLE PLASMA.
1.
E th an o l
fra c tio n a tio n .
2.
P o ly e th y le n e
g lycol
p re c ip ita tio n .
CHARACTERISATION OF THE ALBUMIN.
1.
E le c tro p h o re tic
2.
E l e c t r o p h o r e t i c m ethods,
i)
c e llu lo se
te c h n iq u e s.
a c e ta te
e le c tro p h o re sis
I |)
p o ly a c ry la m id e d is c gel
e le c tro p h o re sis
III)
S D S - p o l y a c r y 1 am ide d i s c
g e ls
Iv )
S D S -po lyacry lam ide s l a b
g e ls
v)
a n a ly tic a l
v i)
p re p a ra tiv e
u ii)
u iii)
ix )
iso e le c tric
fo cu sin g
22
25
fo cu sin g
colum n e x c l u s i o n c h r o m a t o g r a p h y
26
crossed
27
im m unoelec t r o p h o r e s i s
plasm a t o t a l
x)
iso e le c tric
p ro te in
d e te rm in a tio n
p r o d u c t i o n of s p e c i f i c
a n tib o d ie s
31
31
33
RESULTS.
ISOLATION OF ALBUMIN.
33
EXPERIMENTAL MANIPULATION.
33
A.
C e llu lo se
a cetate
33
e le c tr p h o re sis.
th e c h a r a c t e r i s t i c
p a tte rn .
35
1.
E sta b lish in g
2.
C o m p a r i s o n w i t h human p l a s m a a l b u m i n .
35
3.
C om parison w ith
35
4.
C o m p a r i s o n s b e tw e e n l i o n s o f d i f f e r e n t
5.
I n f lu e n c e of
ti m e from i m m o b i l i s a t i o n
6.
I n f lu e n c e of
im m o b ilisa tio n d ru g s.
7.
In f lu e n c e of a n t i c o a g u l a n t .
40
8.
E ffe c t of s t o r in g .
40
9.
E ffe c t of
o th er s p e c i e s .
le n g th of s e p a r a ti o n
areas.
to sa m p lin g .
tim e.
10 .
E f f e c t of d i f f e r e n t c o n c e n t r a t i o n s of sam p le.
11 .
C o m p a r i s o n b e t w e e n s a m p l e s fro m w i l d a n d c a p t i v e
35
35
35
40
41
an i m a l s ,
41
12.
E f f e c t of f e e d in g .
44
13.
P e rc e n ta g e co m p o sitio n of
B.
P o ly acry lam id e d is c
C.
A n a ly tic a l
gel
iso e le c tric
th e a lb u m in s .
e le c tro p h o re sis .
fo c u sin g .
44
44
49
vli
D.
Column e x c l u s i o n c h r o m a t o g r a p h y .
52
E.
P re p a ra tiv e
j2
F.
SDS-di s c g e l
e le c tro p h o re sis.
54
G.
SD S-slab gel
e le c tro p h o re sis .
54
H.
C rossed
1EF,
Im m u n o e le c tro p h o re sis.
DISCUSSION AND CONCLUSIONS.
54
62
MICROHETEROGENEITY OF ALBUMIN.
62
SYNTHESIS OF ALBUMIN AND ITS AMINO ACID SEQUENCE.
66
ALBUMIN GENETICS.
70
ISOMERIC FORMS OF THE ALBUMIN MOLECULE.
71
ANIMAL STUDIES.
73
THE PRESENT STUDY.
76
REFERENCES.
78
1.
Q u a l i t y c o n t r o l of t h e i s o l a t i o n p r o c e d u r e
34
2a. Cellulose ac etate electro p ho retic separation
36
2 b . D e n s i t o m e t r i c a n a l y s i s of e l e c t r o p h o r e t o g r a m
37
3.
Compar ison bet ween human and l i o n a l b u m i n s e p a r a t e d on c e l l u l o s e
acetate
38
4.
Compar i son among v a r i o u s d i f f e r e n t s p e c i e s a f t e r c e l l u l o s e
acetate electrophoresis
39
5.
The e f f e c t of s e p a r a t i o n ti me on c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s 4 2
6.
The e f f e c t o f sample d i l u t i o n on s u b s e q u e n t c e l l u l o s e a c e t a t e
electrophoresis
7.
The e f f e c t of f e e d i n g s t a t u s on e l e c t r o p h o r e t i c s e p a r a t i o n p a t t e r n :
a) overfed
45
b) n o r m a l l y f e d
46
c) starved
47
43
8 a . The e f f e c t o f d i f f e r e n t g e l c o n c e n t r a t i o n s on d i s c g e l
electrophoresis
43
8 b. S e p a r a t i o n of l i o n al bumi n by p o l y a c r y l a m i d e d i s c g e l
electrophoresis
50
9.
10.
Albumin z on e s o b t a i n e d by i s o e l e c t r i c f o c u s i n g
51
I s o e l e c t r i c f o c u s i n g of l i o n plasma
53
55
11.
SDS P o l y a c r y l a m i d e d i s c g e l e l e c t r o p h o r e s i s
12.
C r o s s e d I m m u n o e l e c t r o p h o r e s i s of human al bu mi n a g a i n s t a n t i - h u m a n 57
al bumi n a n t i b o d y ( a g a r o s e / a g a r o s e )
13.
C r o s s e d I m m u n o e l e c t r o p h o r e s i s of l i o n al bumi n a g a i n s t a n t i - h u m a n
al bu mi n a n t i b o d y ( a g a r o s e / a g a r o s e )
14.
C r o s s e d i m m u n i e l e c t r o p h o r e s i s of human al bu mi n a g a i n s t a n t i - h u m a n
al bu mi n a n t i b o d y ( d i s c g e l / a g a r o s e )
58
15.
C r o s s e d I m m u n o e l e c t r o p h o r e s i s of l i o n al bu mi n a g a i n s t a n t i - h u m a n
al bu mi n a n t i b o d y ( d i s c g e l / a g a r o s e )
57
58
16.
C r o s s e d I m m u n o e l e c t r o p h o r e s i s of human al bumi n a g a i n s t a n t i - h u m a n
59
a l bu mi n a n t i b o d y (IEF / a g a r o s e )
17.
C r o s s e d I m m u n o e l e c t r o p h o r e s i s o f l i o n al bu mi n a g a i n s t a n t i - h u m a n
a l bumin a n t i b o d y (IEF / a g a r o s e )
59
C r o s s e d I m m u n o e l e c t r o p h o r e s i s of human al bumi n a g a i n s t a n t i - l i o n
60
C r o s s e d I m m u n o e l e c t r o p h o r e s i s of l i o n al bumi n a g a i n s t a n t i - l i o n
60
18.
19.
\
1
1
During routine in v e s tig a tio n o t plasma sam ples of a v ariety of wild animal
s p e c i e s a d is tin ctly d if fe re n t albumin p a tt e r n was observed in lion plasma
p r o t e in an aly sis. These differe nces were in v estig ated in th i s study. Since th ese
c h a r a c t e r i s ti c s rely on albumin behaviour under d ifferen t conditions it is
n e c e s s a ry to d is c u ss the influence th a t a lte re d environments have on the
p r o p e r tie s of the albumin molecules.
A.
SOLUBILITY IN SOLUTION
Albumin (named a f t e r coagulated egg alb u m n , albus being white in Latin)
p o s s e s s e s ce rta in cha ra c te ristic p ro p ertie s which may be utilized in i t s
is o la t io n and p urification. I t is Known to be an acidic and soluble but stab le
p ro te in . The molecule, in the presence of sta b iliz e rs (such a s caprylic acid),
may w ithsta nd 60°C for 10 hours, a procedure routinely executed to rid clinical
albumin p re p a r a tio n s of h e p a ti ti s B virus. At pH 1 or 2 the molecule elo ng ates
re v e r s ib ly to the so called expanded form (F o ster, 1977). Albumin is relatively
unharmed a t pH 9 to pH 11 but may undergo de naturatio n or disulfide interchange
under conditions of high pH (WalleviK, 1976a). The behaviour of albumin in an
aqueous environment is dependent on the character of the resid u e s attached to
th e alp ha-carbon of each monomeric amino acid unit, since hydrophooic resid u es
will be entropically driven to in te ra c t with one another in order to minimize
w a te r contact w hereas iomzable and polar re s id u e s will in te ra c t with the w ater
so lv e n t. The three-dim ensio nal stru ctu re of albumin is the re fo re a reflection
of th e interac tion betw een amino acid monomeric units (of backbone p ep tid es and
side chain r e n d u e s ) with one another and with the solvent (Lumry and Biltonen,
2.
1969). Solubility of pro te in in a given solvent is determined by the balance
b etw e en p ro te in - p r o te i n and p ro te in -s o lv e n t in te ra c tio n s. Since ail
01
dtein
is o la t io n methods involve some measure of a lte re d solubility i t is e ss e n tia l to
summarize the p a ra m e te rs of p rotein solubility.
1 Molecular size of the p r o t e in
Both size and shape of a p ro trin mol»;ule, described by i t s StoKes radius, w.ll
determ ine i t s exclude (effective) volume (Tan ord, 1961) and it is generally
recogn.sed th a t o ro te m s with large exclude volumes are le s s soluble than
sm aller macromolecules.
2 Amino acid composition, sequence and conformation of the pro tein
The r a t io of polar to non-polar re s id u e s, th eir d is trib u tio n (in clu sters) along
the polypeptide chain and th eir accessibility to the solvent will influence the
p ro te in so lu b ility . Apart from th es e inherent fa c to rs, combination with oth er
n o n -p r o te in su b s tan ces, of which albumin is known to a t t r r c t and bind many, will
als o a ff e c t solu bility . These substances may be covalently linked, ionically
complexed or hydrophobically bound by var der Waals forces to the protein. The
solu bility of th e s e ligands may, for example, explain the d if fe re n t solu b ilities
of glycoprotein and lipoproteins in aqueous solution.
3 Charge of the protein
Molecules of the same charge will repel one another in addition to interacting
with dipolar w a te r molecules and are thus more soluble in w a ter solution th an
uncharged molecules. The charge ot a protein is determined by
a) to t a l number of lonizable re s ic u e s j b ) th eir accessibility to the so lven t,c)
the pH of the solution and d) the dissociation c o n s ta n ts of the lonizable groups
which are, in turn, determined by the chemical composition of the groups
th e m se lv e s, of th e i r neighbouring groups and of th e solvent i s well as by the
ionic s tre n g th of th e solvent and the tem perature of the solution (Edsall and
Wyman, 1958), The n et charge of protein in s o lu tio n may th e r e fo re be
manipulated by affecting changes in the so lv ent, like altering the pH. The pH
of minimum solubility of a p ro te in implies t h a t the net charge of each molecule
is zero at t h a t point, which prom otes the ag greg atio n of the molecules. T his is
called the is o electric pH. Binding of simple anio ns or simple cations will
re s p e c tiv e ly d ecrease or increase *he isoe lectric pH but the binding of m etals
will have an e ff e c t th a t is dependent on the specific method of binding and i t s
r e s u l t a n t a lt e r a t io n of the n e t charge (Edsall and Wyman, 1953). The addition
of la r g e r ionic organic s u b s ta n c e s will r e s u lt in stru ctu ra l changes to the
p r o t e in with r e s u l t a n t a lte ra tio n of protein solu bility which will overshadow
th e a lt e r a t io n caused by the change in net charge.
4 Solvent d ielectric constant
This is a measure of dipole moment and molecular polarizability of the medium.
In a so lven t of high dielectric constant (eg.water) dissociation of e le c tro ly te s
i s promoted but in so lv en ts with low dielectric c o n stan ts (eg.ethanol)
solu bility is d e c e a s e d (Frigerio and H ernn ger, 1962). Since albumin is known
to be stab le in th e presence of such organic so lv e n ts, th is
ecomes a viable
option for p recip itatin g albumin.
5 Ionic stren g th
Low concentrations of ions have a solubility e f f e c t on pro te in s ("salting in")
and, conversely, high concentrations have a precipitating e f f e c t ("salting
out"). In an aqueous solution (high dielectric constant) the salting out e f f e c t
dom inates while saltin g in would be g reater in media of low dielectric c o n sta n t
(Ki kwood,1943). The salting out potential of various s a l t s is strongly
dependent on th e specific cationic and anionic composition of the s a l t
(H ofm eister, 1888). These ion e f f e c ts appear to be re la te d to ion size, ion
charge den sity and ion polan za bility which all will influence io n -so lv en t as
w ell a s lo n - p ro te in in te ra c tio n s. In t h i s way ions may act as conformational
s t a b il iz e r s which r e s u l t in increased helix content (eg.sulphate) and thu s will
de cre ase p ro te in so lu bility or they may act as d e n a tu r a n ts which r e s u lt in the
unfolded coil s t a t e (eg.thiocyanate) and th us will in crease p rotein solubility
(Long and McDevit,1952). In comparing the efficiency of various s a l t s in the
s a ltin g out p rocess it is e ss e n tia l to observe both the completion of
p rec ip ita tio n of the f i r s t protein fraction as an indicator as well as the
increment of s a l t required for each subsequent fractio n. On th i s second b a sis
of comparison i t was found th a t the quantity of chloride required for each
p re c ip itatio n is of th e order of five tim es the in itia l concentration for the
corresponding su lp h ate (Howe, 1923). When comparing th e various sulphate s a l t s
i t was found t h a t sodium and potassium are equally effe c tiv e , followed by
magnesium and th en lithium. These d ifferences seem to be a function of the
degree of hydration of the ions of the s a l t solution with highly hydrated ions
being more eff e c tiv e than le s s hydrated ions.
6 Temperature
In s a l t - f r e e , low ionic stren g th and e th a n o l-w a te r so lu tion s the solubility of
th e p rotein is endothermic; it shows a positive tem pe ratu re coefficient of
solu bility. The specific protein will determine w heth er the l a t t e r is positive
or negative in concentrated ion solu tio ns (A.Uerton, Ward and Fevold, 1945).
B.
EXPERIMENTAL MANIPULATION OF PROTEINS
Induced molecular conformational changes may be affe cted by tem perature,
p r e s s u r e , pH, organic so lv e n ts or n e u tra l s a l t s . As a gen era' rule
■fractionation i s conducted under conditions of a) using the low est possible
te m p e ra t u re , e sp ecially when using organic so lv en ts, b) re s tric tin g the pH as
n e a r to n e u tr a li ty as possible and c) using s a l t s and r e a g e n t s conservatively
(Tanford, 1970). A part from th es e general principles some specific precautions
have come to lig h t in the recent p a st.
P r o t e in s are su rfa c e active and are s tru c tu rally affected by a ir -w a te r or
s o l id - w a te r i n t e r f a c e s . Introduction of a ir into p rotein solutions and the
r e s u l t a n t g e n e ra tio n of foam should be avoided by careful handling of the
sam p les (Bull, 1971).
Pumps and s t i r r e r s could lead to sh ea r damage of the molecules and
ould be
carefully employed in the handling p ro cess (Charm, 1971).
The techniques employed in the addition of re a g e n ts to p rotein solutions require
carefu l co n sid era tio n since local e x c e ss e s may lead to d ena turatio n of the
p ro te in . Liquid, r a t h e r than solid re a g e n ts , added beneath the surface of the
s o lu tion in a multidirectional spray fashion seems to be an accepted method.
The more gradual addition of re a g e n ts by d ialy sis has also been suggested
(McMeeKin, 1939).
When employing one of the sep aratio n techniques it is im portant to determine and
adhere to the e x a ct equilibration +ime of each prec ipitatio n ste p since the
d egree of aging o 1 solutions a f t e r addition of re a g e n ts will a lt e r the
efficiency of th e subsequent p recipitation (Watt, 1970). Regarding the
co ncentration of th e protein solution due for frac t.o n a tio n it has been
su gg ested t h a t a reduced to t a l p rotein concentration will increase the resolving
power of any method. On the o ther hand, p rotein is much more stable
6.
s tr u c tu r a ll y in a c oncentrated solu tion or in the solid s t a t e because in dilute
s o lu ti o n s the molecules a re more exposed to an environment of high dielectric
c o n s t a n t and may be denatured and form insoluble p re c ip ita te s. There th erefo re
e x i s t s an optimum dilution minimumizing the above e ff e c ts of extrem es of
co n c en tra tio n (Cohn, 1925).
O xidative p ro c e s se s could also a lt e r th e quality of the re s u lta n t precipitate
and in some la b o r a to r ie s it has become custom to include metal chelato rs in the
p ro te in so lu tio n s to p reven t th is oxidation (Pennell, 1960; Ray, Davisson and
C repsi, 1954).
Plasma contains sig nificant levels of proteolytic enzymes in active or inactive
p re cu rso r forms. In order to avoid th e i r action p ro te ase inh ib ito rs may be
added to the s t a r t i n g plasma or to the D iffers employed. Since metal ions
a c tiv a te some of t h e s e enzymes, chela if"? will again be effec tive in preventing
activ a tio n . O peration a t the lowest possible tem perature also reduces enzyme
activ ity .
P ro te in solutio ns are generally excellent media for bacterial growth and
p re cau tio n s to p re v e n t contamination like using a closed sy ste m , working at low
te m p e ra t u re s and adding bacteriocidal or b a c te rio s ta tic ag en ts to the solution
will reduce th i s .
Keeping th e s e o p eration al c o n stra in ts in mind, it is clear th a t no single method
of se p a r a tio n will r e s u l t in pure albumin. Usually a combination of differen t
methods will r e s u l t in the highest yield relativ ely pure albumin. Possible
f r a c tio n a tio n methods are thus based on one of the following principles
a) in te ra c tio n betw een the protein and soluble re a g e n ts or so lv e n ts. This may
c o n s i s t of e it h e r major changes affected to the solvent or of major changes to
th e pro te in i t s e l f caused by the binding of r e a g e n ts ,
b) in te ra c tio n of p ro te in with insoluble media,
c) in te ra c tio n of pro tein with physical fields.
C. APPROACH OF THE PRESENT STUDY
In t h i s study the albumin was f i r s t of all is o la te d from
the lion plasma. This
had to be undertaken in order to ascertain t h a t the peculiar p a tt e r n obtained in
th e p ilo t e xperim ents were indeed due to an unusual albumin configuration. To
t h i s end two d if f e r e n t combinations of methods were u tilise d and th e ir r e s u l t s
compared.
Secondly the albumin was characterised by in v es tig atin g i t s electrophoretic,
chromatographic and antigenic p ro p ertie s. A variety of d if fe re n t methods were
employed for dem onstrating each property.
F or comparative purposes the methods selected were chosen to be c lo s e s t to the
r o u tin e in v e s tig a tio n s regularly performed in clinical and experimental protein
a n a ly s is . Since some r e s u l t s could be a ttrib u ta b le to a specific methodological
ca u se , d if fe re n t methods with similar aims were used to rule out the p ossibility
of ascribing a certain c h a rac teristic to the in v e s tig a te d p ro tein on the b a sis
of one analytical method only.
MATERIALS AND METHODS
Blcod w as obtained by venopuncture from immobilised and a n a e s t h e ti s e d lions of a
range of orig in s all over Southern Africa. Since the method of sampling,
orig in s of donors and handling techniques of the m aterial formed a b a sis fo,
comparison, t h e s e a sp e c ts are dis cussed se p a ra te ly in the section en titled
"R e su lts" .
A. ISOLATION OF ALBUMIN FROM LION WHOLE PLASMA
1. E thanol fra c tio n a tio n (Hao, 1979)
Plasma diluted th reefo ld with isotonic saline, was placed in a w ater bath a t -5
C and the pH w as adjusted to 5,6, using a 0,8M a c e ta te b uffer, pH 4, prepared
from 2,5 tim es diluted glacial acetic acid and 160g/l solution of NaOH in the
r a t io of 4:1 (Cohn, Heyroth and MenKin, 1928).
A fte r s tirr in g for one hour, precooled ( - f o 95% ethanol was added in a thin
s tream fashion (to avoid h e a t production) to a final concentration of 42% (V/V).
The te m p eratu re of the mixture was Kept as close to -5°C as po ssib le but, in any
case, throughout the procedure never exceeded 0°C. A fte r the eth anol addition
the pH was 5.8 and the mixture was continuously s t i r r e d for a t l e a s t one further
hour. This c o n sta n t low tem perature was achieved by using a circulating bath
filled with glycol into which the mixture was submerged. The bath was s e t up
over a magnetic s t i r r e r .
C entrifu g atio n for one hour at - f c was performed in a Sorvall re f rig e r a te d
cen trifug e operating at 12000 x g. A fterw ards the precipitate w as discarded and
the s u p e r n a ta n t used to r fu rth e r fraction atio n. The pH of the l a t t e r was
ad ju s te d to 4.8 using ace ta te b uffer and, a f t e r stirr in g for one hour the
mixture w as allowed to stand *or a t le a s t th ree hours (Cohn e t al.,
1946).
A fte r t h i s ageing the mixture was again centrifuged for one hour. The
r e s u l t a n t p re c ip ita te was similar to Cohn fractio n V in tex tu re and was the
albumin p a s t e used fu r th e r. The p a s t e was re c o n s titu te d in d is tille d w ate r and
then lyophilized. The powder product was sto re d and subsequently was dissolved
fo r pu rity checks and fu rth e r ana lysis. By using ele ctrophoretic techniques the
s e p a r a ti o n efficiency of the procedure was a s s e s s e d in each s t e p and by using
p ro te in e stim atio n techniques, the yield of the final product was calculated.
This was alw ays higher than 90% and with no electrophoretically d etec table
im p u rities.
2. P olyethelene olvcol p recipitation (Curling e t al., 1977).
The pH of the s t a r t i n g plasma was adjusted to 8.0 by the addition of 1M NaOH.
P olyethelene glycol (PEG) 4000 was made up to a 50% W/V concentration <50g made
up to 100ml) and added to the plasma (which was continuously stirred ) up to a
final co ncentration of 12% W/V.
This mixture was s t ir r e d a t 4°C for one hour and th en centrifuged for 15
m inutes. The s u p e r n a ta n t collected was adAisted to pH 4.6 and solid PEG was
added while s t irr in g , to a final concentration of 25% W/V. A fte r one hour of
s t ir r in g , cen trifu g atio n for 15 minutes yielded a p recipitate which was washed
in 25ml of cold d istille d w ater and again c entrifuged. The final pre cip itate
w as dissolved in 20ml cold d is tille d w ater and the pH adjusted to 7.0.
o
DEAE Sephade:: A-50 was swollen in excess volumes of 1M sodium a c e ta te a t 96 C
fo r two hours and th en tra n s fe r re d to a 0.05M sodium a ceta te acetic acid bu ffer
1 0.
of pH 5.2 and packed in a column (15cm x 1cm). This packed column was
e q u ilib ra te d with two column volumes of the buffer.
The pH of th e l a s t solution was reduced to 6 and i t w as th en applied to the
column in th e r a t io of 5ml solution for each 0.5g dry ion exchanger. The column
w as th en washed with one column volume of b uffer to remove residu al PEG and
unbounci p ro te in . The b uffer pH was lowered to 4.6 with an accompanying increase
of ionic s t r e n g th . These conditions then eluted the albumin.
F rac tio n s of 1ml were collected at a flow rate of 0.5ml/min and th e ir light
absorbance was monitored a t 254nm wavelength. In t h i s way the fra ctio ns curing
a peak of increased absorbance could be collected and sto re d for fu r th e r use.
Quality control was again executed by means of electrophoretic techniques to
exhibit purity of s e p a ra tio n and by p ro tein concentration e stim atio n s for
percentage albumin yield. The former was occasionally quite obscure because of
the dilution of the p rotein but the l a t t e r measured a yield con sisten tly above
80%.
b . c h a r a c t e r i s a t i on o f t h e a l b u m i n .
Since the majority of in v es tig a tio n s into the p ro p e r tie s of albumin involve some
type of electrop ho retic technique in th i s study, it would be appropriate to
d iscu ss the principles of electrophoretic sep aratio n a s they can influence the
conclusions reached about the specific albumin condition studied here.
11.
E lec tro p h o retic techniques
Many biological molecules p o s s e s s ioni:able groups and may th us be made to ex is t
in s o lu tio n cs elec trically charged e n ti ti e s . Molecules with a similar charge
b u t w ith d if fe re n t molecular m asses s till d if fe r on the b a s i s of the charge/,* a ss
r a t i o . The d i f f e r e n t ia l migration seen in such molecules when they are placed
in an elec tric field forms the b a s i s of e lec trop ho resis.
The r a t e of movement of cations to the cathode and anions to the anode is
d eterm ined by th e balance betw een the attra c tin g forces of the electrical field
and the frictio nal and e le c tr o s .a tic e ff e c ts of the movements of charged io ns in
a medium. For movement to take place the sample must be dissolved in a b uffer
so lu tio n and th e medium must be sa tu ra te d with buffer in order for the curren t
to be conducted.
The current is maintained by e le c tro ly sis taking place a t the e lectrod es so
t h a t , » t the cathode, hydroxyl ions and hydrogen are produced and oxygen and
hydrogen ions a t th e anode. The cathodal hydroxyl ions cause increased
d is so c ia tio n of th e weak acid in the buffer with the r e s u l t a n t increase in
unions which will conduct the cu rren t to the anode. There the anions combine
w ith hydrogen ions to reform th e acid while the elec tro ns are refed to the
e le c tric circuit. The sample ions join th ese b u f f e ' ions in th e ir migration
and are s e p a r a te d on the b a sis of th eir electrophoretic mobility. When allowing
t h e s e migrations to take place on an inert supporting medium one causes the ions
to s e p a r a te into d is tin c t zones. The nature o* the supporting medium will
determ ine the in te ra c tio n with the ions being s e p a ra ted and could exploi* the
d iffe re n c e s in ch a rg e /m a s s r a t i o s or retard the sample.
Migration r a t e is influenced by various fa c to rs. The r a t e in crea se s with an
increase in n e t charge of t^e sample ions which, in turn, was determined by the
pH of the sam ple. The r a t e d e creas e s fo r larger molecules because of increased
fric tio n a l and e le c t r o s t a t i c forces. Molecules of the same size but with
d if f e r e n t s h a p e s will migrate at d if fe re n t r a t e s ; fib rou s and globular pro tein s
migrate d if f e r e n t ly . Changes in the elec tric field will also exert an e ff e c t in
th e sen se th a t an in crease in the po ten tial gradient (measured in volts) will
in c re a se the r a t e of migration. Similarly, an increase in current will increase
m igration r a t e . Conversely, any re s is ta n c e exerted by the support medium will
r e t a r d the r a t e of migration. An increase in tem perature will cause increased
mobility of ions as a r e s u lt of evaporation of solvent from the support medium
which will dec rea se r e s is ta n c e . This is , however, overbalanced by the increased
b u ff e r ion c oncentration with a net r e s u lta n t decrease in migration r a t e .
Direct cu rren t was supplied through power packs which deliver e ith e r co n stan t
voltage or c o n sta n t current which makes subsequent comparisons p o ssible. With a
c o n sta n t voltage applied one ob serves a rising cu rren t during elec tro p h o resis
due to a dec rea se in r e s is t a n c e of the medium because of a rise in tem perature.
While th is may not be problematic in low voltage paper elec tro p h o re sis the h eat
ge nerated is n ot e asily d is sip ated with g e ls or cellulose a ceta te and in th ese
conditions c o n s ta n t current should ra t h e r be used. A closed chamber will reduce
evap oration and a cooling system will help reduce overheating. A group of media
running in p a ra lle l will also cause too much heat when run with a c o nstan t
vo ltage. C o n stan t cu rren t is again p re fe rre d but should be increased in
proportion to the number of media used. For absolute reproducable conditions
the voltage drop p er cm length of medium, the current density per cm width of
medium and the te m p era ture should be accurately measured.
The b uffer used has to, f i r s t of all, d issolve the sample so th a t the op erato r
should be wary of sample diffusion, especially if the sample ions are small.
Low ionic s tre n g th b u ff e rs will r e s u lt in a sm aller portion of the current being
carried by the bu ffer ions and, consequently a la rg e r portion carried by the
sample io ns. This will increase the r a t e of migration. I t also cau ses le s s
h e a t production but sample diffusion and lo ss of re s o lu tio n is higher. On the
o th e r hand, high ionic stre n g th b u ffe rs will d ecrease the portion of current
carried by the sample ions and hence reduce the migration r a t e . In addition it
will in c re a se the ov erall current and h e a t production. The eventual choice of
b u ff e r ionic s t r e n g th is a compromise betw een t h e s e two ex trem es.
The pH of the b u ffe r has l i tt le e ff e c t on fully ionised compounds such as
inorganic s a l t s but for organic compounds pH will determine the degree of
ionization. Increasing b uffer pH le v els will cause increased ionization of
organic acids and d ecreased ionization of organic bases; the r e v e r s e applies to
d ecreasing buffer pH lev els. In most electrophoretic sy s te m s there is a
continuous b uffer system over the medium but in certain disc gel sy stem s another
b u ff e r is used in th e gel its e lf; th is is thus a discontinuous bu ffer system .
Regarding the su p p o rt medium, it is usually considered to be re latively in e rt
but n e v e r t h e le s s can in tera ct to some e x ten t with the sample ions and th erefo re
can be employed to manipulate mobility. Sample ions can be adsorbed to the
medium, causing a "tailing" instead of movement in a band shape and thus
reducing ra t e and reso lu tio n of se p a ra tio n . Surface adso rp tion of ions from the
b u ff e r o r the presence of sta tio n a ry carboxyl groups on paper or sulphonate
groups on agar can r e s u l t in a re lativ e charge existing betw een the medium thu s
charged and the w a te r molecules in the b uffer. This g e n e ra te s a motive force
for fixed anions to the anode and r e s u l t s in movement of hydromum ions to the
cathode, carrying along neutral su bsta nce s by so lv ent flow. In th is way so
called elec tro -en d o sm o s is a c c elerates the movement of cation s but r e t a r d s *hat
of anion s. This i n t e r f e r e s with determ inations based on obtaining +ie
14.
is o e le c t ric p o in ts of compounds and for correction purp oses the migration of
n e u tr a l su b s ta n c e s such a s urea or glucose must be measured in the same system .
Gel media, which all have some randomly intertw ined molecular chain s tru c tu re ,
can act like a sie v e . In th is way agar, starch and polyacrylamide gels re ta rd
th e movement of la r g e r molecules which utilize them with more ease .
In view of the numerous v ariatio n s in p a tt e rn obtainable from the same sample
due to exp erim en tal manipulation and the need to r e s t r i c t characterization to
o f t e n used sta n d a rd techniques in order to compare r e s u l t s with published
ro u tin e r e s u l t s a few applications of the above concepts should be considered
fo r e ff e c tiv e characterization.
ELECTROPHORETIC METHODS.
1. Cellulose a c e t a te e lec tro p h o resis
C ellulose a c e t a te s t r i p s have a very homogeneous pore stru c tu r e and allow very
l i t t l e ad so rp tio n of macromolecules. It is l e s s hydrophylic than paper, holds
l e s s b uffer and th erefo re allows a b e tt e r re solu tion in a sh o rter time. This,
however, can lead to heat being produced but, by using a closed chamber, drying
of the medium s t r i p s was avoided. An added advantage is th a t the background can
be i endered tr a n s p a r e n t by tre a tm e n t with a clearing agent followed by heat
drying. I t is th u s generally considered superior to paper and has become very
popular clinically. In th is study tr is - b a rb it a l- s o d iu m -b a rb i ta l buffer (pH 8.8)
w as made up and 100ml b uffer with 0.5ml ethanol used as a w etting solution to
soak th e cellulose a c e ta te s t r ip for at le a s t 10 minutes. A volume of 200ml
b u ffe r was used in the elec tro p h o resis chamber with both sides equally filled.
A f te r the removal of the s t r ip from the soaking solution it was placed on a
s h e e t of a b so rb en t paper. Meanwhile the application block was loaded with
sam p le s and s t a n d a r d s with th e use of a P a s t e u r p ip e tte . The block may be
covered with a lid in betw een loading procedures to prevent evaporation. From
th e r e the t e s t so lu tio n s were picked up by th e applicator which absorbed the
sam p les betw een the two p a ra lle l w ires when th e applicator key was d epressed for
10-15 seconds. This was then used to t r a n s f e r the samples and s ta n d a rd s to the
medium strip which has to be lightly b lo tted with another s h e e t of absorbent
p aper and then placed in the chamber before loading. The ele c tro de s were placed
o ver the chamber in th eir fixed position and plugged into a Vokham power pack on
which the voltage was adjusted to 225V. E lectro p h o res is was allowed to proceed
for 20 minutes. Immediately a f t e r switching o ff the power supply the s t r ip was
removed from th e chamber and floated on top of the sta
nn (0,5%
Ponceau S in 7,5% aqueous trichloroacetic acid). Wner,
npletely wet i+
was submerged and stained for 10 minutes. Using forceps, the s t r ip was removed
from the stain and placed in a 5% acetic acid rinsing solution which was then
a g ita te d to remove excess s ta in . This rinsing procedure was th en rep eated in a
second and th ird acetic acid r i m . ' for a to t a l time of about 10 minutes until a
clear background w as obtained. The s t r ip was th en removed from the final rinse
tr a y and allowed to drip off th e excess rinse solution before being put in Sepra
(R)
C lear clearing solu tio n for 5 minutes. A fte r th is the s t r i p was placed
lengthwise on a g l a s s slide and a second g la s s slide edge was pulled over th is
at 45°angles t c remove all the air bubbles trap ped underneath the s t r ip . The
fr e e edges were th e n folded over and the slide was placed in an oven preheated
to 80° 90°C and l e f t for 20 minutes to produce a completely tr a n s p a r e n t and dry
background. A f te r cooling down the slide was cleaned with w ater and could then
be sto red for f u r th e r analy sis la te r. D ensitometric (Gelman scanning
computerised d ensito m eter) analysis could provide information regarding the
re la tiv e contribution of the various protein frac tion s sep arated on the paper).
16.
2.
Polvacrvlamide disc gel elec trop ho re sis
The following four so lu tio n s were made up:
1. 20g acrylamide and
0,8g N'N' methylene bisacrylamide
in 100ml d istille d water
2. l,6g or 1,34ml dime*hylaminopropiomtrate and
40ml stock b u ffer (below)
in 100ml d istille d w ater
3. Stock buffer: 3g T n s (methylamine) and
14,4g glycine in
MOml distilled w ater
(pH should be 8,5)
Working buffer: Stock buffer diluted 10-fold
4. 0,5% ammonium persulphate
These so lu tio n s can be sto red in dark b o tt le s in the fridge. One end of the
special 2ml g la s s tu b e s was sealed with parafilm or a close fitting plug. Eight
of t h e s e tu b es were placed in a gel casting stand and kept v ertical. 5ml of
each o f th e four so lu tio n s was mixed and then sucked up in a large syringe. The
l a t t e r w as used to fill each vuhe for about 75% of i t s length and very carefully
about two drops of d is tille d w ater was lowered onto the above mixture in each
tube to s e a l it o ff and cause solidifying. After about 15 minutes th e tubes
w ere t r a n s f e r r e d to the discontinuous electrophoretic chamber with the bottom
chamber fil
d with working bu ffer to cover the e lec tro d e su fficien tly. Tube*
w ere th e n unplugged a t the bottom and the w ater lay er a t the top removed before
being lowered in to the buffer, ensuring th a t no a ir bubbles are trapped
u n d ern ea th each tub e. The upper chamber was th en a ls o filled with working
b u ff e r and. using a syringe, air trapp ed on top of th e g e ls was displaced by
b u f f e r . The top elec trode was lowered into the b u ff e r and should be completely
subm erged. The e lec tro d es were then connected to th e Vokham power pack so th a t
the n egativ e terminal was i t the to p and the p o sitive a t the bottom. A con stan t
v o ltag e of 20V w as appli-d for 1-2 minutes. On a s h e e t of parafilm ^til of
sample was used to dissolve a few c ry s ta ls of su cro se to weigh the sample down
o nto th e gel surface when placed under the level of b u ffe r. Blank tu b es could
be loaded with pro te in solution sta in e d with bromophenol blue to secure a
visibly migrating fron t in order to judge range of migration.
A f te r applying all the samples on the top surface of th e gels and replacing the
top elec tro de, the power pack was switched on to supply 90V for 20 minutes and
th e n 160V for again 20 minutes to th e chamber. The sta in e d protein could be
visually checked to ensure adequate distance of movement. A fter removal of the
e le c tr o d e s the tu b e s were removed from the chamber and, using a long needle and
syring e to introduce d is tilled w ater between the gel and inner g la s s surface to
lo o sen the g els, they were removed from the g la s s tu b e s . Gels were then placed
in i r iividually numbered compartments of a stainin g rack and stain ed with 3g/l
amido black in 5% acetic .
for a t le a s t 30 minutes. D estainm g could be
performed electrophoretically in th e Shandon tr a n s v e r s e gel d e sta in e r by placing
th e sta in e d gels in betw een two pads in a d e s ta in frame and firmly wedding it
in to the centre of the d estainm g chamber. Acetic acid (107.) was introduced on
one sid e of the gel stack and allowed to flow t r o u g h the pads. The power pack
was connected to th e d estaim ng chamber with the negative electrode to the clear
side and the p o sitiv e electrode to the side with stain ed acetic acid. Constant
c u rr e n t of 0,2A w as applied for about 2 hours and then the gels were removed
from th e holder and sto re d in 5% acetic acid. Destaining may also be
accomplished by d iffu sio n , by leaving the gels for a day or more in 10% acetic
acid while being s t i r r e d continuously. This, however, was very time-consuming.
Modification of pore sizes m polyacrylamide gels
P o ro s ity of th e s e g e ls is determined by the re la tiv e proportion of acrylamide
monomer to cross-lin kin g agent. The to tal mass per volume acrylamide and
cro ss-linkin g a g e n t is usually e x pressed as a p ercentage. In th is way it is
p o s s ib le to have g e ls ranging from 3% to 30% which yield corresponding pore
s i z e s of 0,5nm to 0,2nm in d ia m e te r.
With th i s in mind th e following percentage gels were prepared:
5%
- 20g acrylamide
0,8g NN Methylene bisacrylamide
7,5% - 30g acrylamide
l,2g NN Methylene bisacrylamide
8,5% - 34g acrylamide
10% - 40g acrylamide
3. SDS - polyacrylamide disc gel
Fairbanks, |971)
T h is variatio n on the disc gel elec tro ph oresis enables one to sep arate sam ples
on th e b asis o( molecular mass alone. In th is way c alib ratio n curves indicate a
li n e a r rela tio n sh ip betw een migration distance and the leg of the molecular mass
of the protein sam ples.
19
In th i s modification th e sam ples are given the same ch arg e /m a s s ra t io by
solubilizing i t in th e following solution:
10g/l sodium dodecyl sulphate (SDS)
lOOg/1 sucrose
lOmM
T n s - HC1 (pH S)
1 mM EDTA (pH 8)
0 , J g / l m ercaptoethanol
lOmg/1 Py.-omn - Y (tracking dye)
This pro cess was carried out a t 100°C for 5 minutes. The
mercaptoethanol
breaks the disulphide bonds and the SDS a c ts as a d e te r g e n t to cause the
solubilization and a tt a c h e s an ionic group a t regular in te rv a ls.
Stock solutio.
1
Concentrated acrylamide bisacrylamide
acrylamide
40g
NiN Methylene bisacrylamide l,5g
in 100ml d is tille d w a te r
2
B u ffer (pH 7,4)
1.0 M T n s-H C l
2.0 M sodium a c e t a te
in 100ml d is tille d w ate r
(adjusted with acetic acid to pH 7,4)
Gels were th en p repared a s follows:
Conc.acrylamide bisacrylamide
B u ffer
5ml
7ml
Water
28ml
1,5% ammonium persulphate
0,5% TEMED
5ml
2,5ml
G la s s t u b e s were u s e f t : contain the individual g e ls and th e s e were gently
o verlaid with 0,1% SDS
0,15% ammonium persulphate
0,05% TEMED
and l e f t for th irty minutes at room tem perature to polymerize.
Running b uffer w as made up as follows:
Gel b u ff e r
100ml
20% SDS
50ml
D istilled w ater 850ml
The g e ls were l e f t in the chambers with the running b u ff e r overnight in order to
prom ote eq uilibratio n.
E le c tro p h o re s is took place a t a p oten tia l difference of 50V/cm and until the
tracking dye h a s migrated about 8cm. A fte r removal of th e gels from the glass
tu b e s they were placed in s l o tt e d t e s t tu b es suspended in a beaker containing
25% is opropranolol and 10% acetic acid which w as ueing s t i r r e d continuously fotw elve hours.
Staining took place overnight in 10% isopropanol and 10% acetic acid containing
0,025% (W/V) Coomassie Blue 250. D estaim ng was executed in the same way as
normal disc g e ls by ele c tro ph oresis or diffusion.
21.
4. s n s Pniyarrylamide slab gels
Slab g e ls were prepared in a commercially a vailab l- casting frame so th a t the
gel w as poured in the same vertical p o sitio n th a t i t was used in, making
t r a n s f e r of the gel u nnecessary.
Samples were prepared according to a modified method (Laemmli, 1970) where the
final sample solutio n contains
0,0625 M T n s -H C l (pH 6,8)
2% SDS
107. Glycerol
57.
2 -m erca pto eth ano l
and,for a marker sample,also
0,0017. bromophenol blue.
T h ese sam ples so lu tio n s were then immersed in boiling w ate r for 1,5 minutes to
en su re complete d is so ciatio n before q u a n titie s of 0,3 ml were loaded into the
sample hollows made in to the gel by applying a comb to the unpolymerized
s u r fa c e .
The gel co n sisted of a sep aratin g portion made up of 87. acrylamide and NN
bism ethylene acrylamide with a final concentration of 0,375 M Tris-H Cl (pH 8.8)
and 0,17. SDS a s well a s a stacking gel made up of 37. acrylamide and NN
bism ethylene acrylamide with a final concentration of 0,125 M Tris-HCl (pH 6.8)
and 0,17. SDS. Both g e ls were polymerized in turn by the addition of 0,0257.
te t r a - m e th y l-e th y le n e -d ia m in e (TEMED) and ammonium persulp hate .
The elec trode b u ffer (pH 8.3) contained 0,25 M T n s and 0,192 M glycine and 0,17.
SDS. Cooling of th e gel during sep ara tio n was necessary and the electro ph oresis
v.'as continued un til the marker has reached the bottom of the gel.
A f te r e le c tro p h o r e s is the p ro te in s were fixed in 50% trichloroacetic acid (TCA'
o v e rn ig h t, stain ed fo r one hour a t 37°C with 0,1% Coomassie b rillian t blue made
up in fre sh ly prepared 50% TCA. Subsequently i t was d iffusio n - d estained in 7%
acetic acid.
5. ANALYTICAL ISOELECTRIC FOCUSING (!& .& )
Gel p rep aratio n
0,3g a g a r o s e - I E F
3,eg sorbitol
29ml distilled w ater
is mixed in a 50 ml flask and heated on an ele c trically heate d magnetic s t i r r e r
u n ti l th e agarose is dissolved.
Mould prep aratio n
A levelling casting p k tf o r m w as adjusted to the horizontal and 2 ml of w a te r is
poured onto it. A $t f i t of OelBond, with i t s hydrophilic su rface upwards was
placed on the tab) i, being handled by i t s edges only. (The hydrophilic side can
be id en tifie d a? the side on which a water drop will "spread" and not "bead".
E x c e s s air b u ib l e s and water were removed from underneath the film by rolling it
f l a t with t ro ller or g la s s rod and the ex pressed w ater is removed from around
th e e dges with ti s s u e paper.
The casting frame was th en fa s ten ed over the film and held on by spring clips.
With the use of a hairdryer the film was then thoroughly prewarmed.
G el m oulding
o
Once all the ag aro se was d issolved, the mixture was allowed to cool to about 75
C b efore 1,9 ml of the c a rrie r ampholyte, Fharm alyte, was added. The mixture
was then s t i r r e d and quickly poured into the mould, ensuring th a t the solution
flowed into all co rn e rs and t h a t no air bubbles e x is te d in the gel (a hot needle
will "pop" th e s e before the gel s e ts).
A f te r 10-15 minutes the gel had s e t and could be se p a ra te d from the frame by
pulling a scalpel around the edges. Having removed the casting frame, the film
with gel could be lifte d o ff the casting platform . Subsequently gel was allowed
t o harden at 4°C for one hour or overnight a t room tem perature. During th is it
should alw ays be sto re d in an air tig h t container with moist tis s u e paper to
avoid drying out.
S ep aratio n
A small q u an tity (2 ml) of w ater is placed on the cooling platform (Pharmacia
FEE 3000) so t h a t , when placing the gel-carrying film, there is a thin layer
underneath over th e whole surface,
The elec trode s t r i p s were then each soaked in e it h e r 1M sodium hydroxide or 0,05
M sulphuric acid and subsequently b lo tted for one minute to eliminate excess
solu tion b efore being placed a t the cathode and anode respe< *ively.
Plasma sam ples of 20yul were applied to the gel by means of paper applicator
s t r i p s . The e le c tro d e s in the lid should be adjusted to ensure contact with he
24.
electrode s t r i p s when the gel is covered.
The c o n s t a n t power supply (Pharmacia ECPS 3000/150) was then s e t to deliver a
maximum of 15 W and 1500 V w ith unlimited curren t for one and a h alf hours.
The cooVng platform was connected to a circulating w a te r bath which keeps the
tem p era tu re of th e gel environment a t 10°C. Sample applicator s t r i p s could be
removed a f t e r 45 minutes.
Fixing and sta in in g
Immediately a f t e r completion of the run the gel was immersed in fixing solution
for 30 m inutes. This solution consisted of:
5% Sulphosalicylic acid
10% Trichloroacetic acid
in d is tille d w ater
The gel w as now rinsed in two washings of destaining solution, each for 15
minutes. This so lutio n con sisted of:
35% ethanol
10% acetic acid
in d is tille d w ater
The gel w as th e n dried by puttin g three la y e r s of f i l t e r paper over it , followed
by g l a s s p la te and a weight of about 1 kg. A fter about 15 minutes all th is was
removed and the gel dried with a hairdryer.
The dried gel w as then placed in staining solution for 5-10 mminutes. This
solution co n siste d of 0,2% Coomassie Blue R250 in destaining solution.
Subsequent d e sta in in g should be of 15-30 minute d uratio n, su ffic ien t to obtain a
d e a r background. The gel film w as then dried with a h aird ry er and could be
s t o r e d as a perm anen t recoro.
6. PREPARATIVE ISOELECTRIC FOCUSING (lEF)
A part from the analytical s e p a ra tio n by is oelectric focusing, the method can
a ls o be performed or, a p rep arativ e scale. The medium chosen in th is study was
Sephadex - IE F , a specially prepared Sephadex G - 75 for use in p re p a ra tiv e IEF.
Sephadex <15g) w as swollen in 225 ml d is tiled w ater and 12 ml of the c a rrie r
ampholyte, Phar malyte, was then added before the gel is dea erated .
The special gel trough (Pharmacia) was placed on a horizontal casting platform ,
supported by th e g la s s plate su p po rts. The gel slurry was poured into th e
trough and with the help of the gel spread er, it was evenly d is trib u te d . The
opening of th e Sephadex - IEF b o ttle was covered with a fine gauze held in place
w ith an e la s ti c band and additional Sephadex was sprinkled over the e n tire
surface u n til th e gel remained immobile when tilte d by 45 d egrees. The gel
consistency would th en be .onsidered correct.
With the use of a s t r i p from the fra c tio n a to r grid a slice of 1 cm wide was
reno veu from each end of the gel trough and the electrode s t r i p s were soaked in
0,1 M sulphuric acid for the anodal electrode and 1 M ethylenediamine fo r the
cathodal electrod e and placed in th ese spaces. Distilled w ater (5 ml),
containing 1% d e te r g e n t, was placed on the cooling p la te under the gel, avoiding
i i r bubbles being trapped th e re . The cooling circulating w ater bath was th e n
sw itched on a t a te m p era ture o ' 10°C.
P refocusing of 45 minutes at 8 W is recommended before sam ples can be loaded.
The l a t t e r was achieved by in s e rtin g the applicator spacer into the gel and
scooping out all th e s e p arate d gel into a beaker where it was mixed with the
sam ple before being returned to the applicator space with the aid of a syringe.
The power pack w as s e t to deliver 50 W for 6 hours and switched on about 10
m in utes a f t e r sample application to allow for equilibration.
The se p a r a te d molecules could be detec ted with a replica technique, involving
ro lling over the gel surface an exactly fittin g s h e e t of Whatman 1 MM f i l t e r
p a p e r and leaving i t in position for 2 minutes. A fte r drying, the paper was
fixed and sta in e d in th e above solu tio ns. From t h i s replica s e p arated zones
w ere iden tified with the help of th e fraction*
vn grid which could th en be
introduced into th e gel and, by in serting cutting s t r i p s , were cut out of the
gel p la te and scooped out. This gel was th en re su spen de d in b uffer in a
su ita b ly sized column from which the substance is o la te d could be eluted with one
column volume o, bu ffer.
7. Column exclusion chromatography
In t h i s experiment commercial columns (Pharmacia) were used. Sephadex G 200
s u p erfin e gel p a r t ic l e s a"e swollen and equilibrated for th ree days in the
b u f f e r solution consisting of 0,1 M Trls and 0,5 M NiCl in distille d w ater with
th e pH adjusted to 8.5 with HC1.
A 2 ml sample o t albumin reco n s titu te d in bu ffer solution <40 g/1) was added to
27.
th e top gel su rfa c e by removing all the solvent above the gel column and then
applying the sample directly onto the top surface w itho ut ♦he gel running dry.
B u ffe r was th e n carefuly added to t h i s layer to a h e ig h t of about 10 cm before
th e re s e rv o ir w a s again connected up. This could e a s ily be done and thu s
obviated the need to weigh the sample solution down with 1% sucrose dissolved in
i t or the need for d irect application by means of a syringe in serted through the
s o lv e n t above th e gel su rface.
Movement of so lv en t through the column was aided by a p e rista ltic pump
(Pharmacia P - 1> s e t to provide a solv en t flow of 5 ml/hour. Only t h i s one
so lv en t w as used in eluting the molecules, mating t h i s an is ocratic method o,
s e p a r a tio n in c o n tr a s t to the g radient elution t h a t r e l i e s on a continuously
changing pH of the solvent.
A single path u ltra v io le t monitor (Pharmacia) was used to monitor the passage of
th e p ro te in s since they absorb the ligh t
.ally a t 254nm wavelength.
3. Crossed Immuno elec trop ho re sis (WeeKe, 1?.73)
This technique combines th e use of electrophoretic sep aratio n of pro tein s in
agarose gels followed by ele c tro p h o r e sis perpendicular to th is in to an
antibody-containing gel. Immunological behaviour o f each se p a ra te d protein is
th u s dem o nstrated.
The following stock b u ffer was made up
44,3g Tris
22,4g Barbitone
0,53g Ca - lacta te
28.
0,65g Na
azide
made up to 11 with d is tille d w ater (pH 8,6,.
This w a s d iluted 1 * " with d is tilled w ater for th e ex perim ents.
With the use of t h i s bu ffer a 1% agarose solution was made and fully dissolved
by ca refully h eating the mixture to just boiling point and th en measuring out
with a p ip e t te 15 ml which was run over the preheated (hair dryer) surface of a
90 x 110 x 1,5 mm g la s s plate which was held horizontal on a gel casting
p latfo rm . Avoiding the formation of air bubbles and spreading the gel over the
e n ti r e su rface should r e s u l t in a uniform homogeneous gel of about 1,5 mm in
thick ness.
A f te r congelation of th " agarose a well (2,5 mm in diameter) was cut in the one
corner of th e gel and the gel was placed on a cooling platform with ta p w ater
running through. The bu ffer tanks were filled with the diluted b u ffer and
connected with the g?' bv means of th ree f i lte r paper wicks. The gel was turned
so t h a t the direction of migration during e le c tro p h o re sis was 'w ay from the well
along the long sid e of the gel. The anode was th u s connected to th e far side
and th e cathode t c the well side of the gel. 5yul of sample was measured into
th e well, the ap p a ra tu s was covered and e lec tro p h o resis a t 350 V (constant
voltage) was performed for 45 minutes. A marker drop of bromophenol blue could
be placed on the gel and run along the opposite side a s the sample.
A f te r completion of th is the top tw o -th ird s of the gel (60 x 110 mm gel) was cut
o ff from the 30 x 110 mm s t r i p in which the sample ran and the former was peeled
off th e g la s s p la te which was then returned to the casting platform .
The I t -g a r o s e was again used by heating the mixture to just boiling point and
th en allowing it to cool off to about 55°C. At th is tem perature 12 ml of gel
w as carefully mixed with the commercially ob tain ed antibody (anti human albumin.
DaKo) which was measured out in to t e s t tub es in a w ater bath (SO^C) beforehand
to p r e h e a t . The exact amount of antibody so lution determines the antigen /
an tib o d - ra t io which is crucial fo r the second dimension range of migration. In
th e s e e xperim ents 20yul and 6 0 /jl were resp ectively used in two s e t s of
s e p a r a ti o n s .
The gel was carefuly mixed with the antibody solution in the w ate r bath without
g eneratin g air bubbles. This w as then poureo over the denuded area of g la s s
next to, and overlapping slig htly with, the remaining sam ple-containing gel.
A f te r congelation (10 - 15 minutes) the gel s lid e was tr a n s fe r re d back to the
e le c tro p h o r e s is tank and s e t up to allow m igration out of the f i r s t gel
(cathodal side) into th e antibody containing second gel (anodal side) and the
wicks were placed.
E le c tro p h o re s is was carried out under the same conditions mentioned above but
only fo r 35 - 40 minutes or un til a dye marker sample had run the whole width of
th e p la te .
A f te r completion the gel p late w as removed and covered with saline ana th ree
s h e e t s of f i l t e r paper and a g l a s s slab with about 1kg mass on top. This was
continued for 12 hours but the paper was replaced three times during th a t
period.
Then the p late was dried in a preheated oven a t 90°C for 30 minutes and then
allowed to cool down before i t w as immersed in the following staining solution
for 20 minutes.
30.
5g
Coomissie B rillia nt Blue R250
450ml 96% ethanol
100ml glacial acetic acid
450ml d istilled w ate r
The solu tion w a s le f t standing overnight a f t e r mixing and was th en filtered
b e fo r e use.
A f t e r th is th e gel was d e stain ed three tim es, each time with fresh d estain m g
so lu tio n for a t o t a l time of 15 minutes in a mixture of:
450ml 96% ethanol
100ml glacial acetic acid
450ml d istilled w a te r
The gel w as th e n d n e ^ at 90°C in the oven for 5 - 1 0 minutes.
F u rth e r modifications of t h i s technique would apply d if fe re n t methods to the
f i r s t dimension of sep a ra tio n . In th is experiment is o e le c tric focusing,
polyacrylamide disc gels and SDS-polyacrylamide disc g e ls were used for the
in i ti a l s e p a r a tio n .
F o r id e n tif ic a tio n in this s t e p the polyacrylamide g els were cut lengthwise
(b es t perform ed when frozen) and the gel bond of IEF could be sp lit. In both
c a s e s the o th e r half was th e n be stained normally. Before pressing a f t e r the
second dim ension elec tro p h o resis the half gels of polyacrylamide had to be
removed from th e glass but the IEF s t r ip could remain and be p ressed and dried
w ith the a g a ro s e .
9. P lasm a to ta l p ro te in determination
T h ese e s tim a tio n s were done by using the method of Lowry e t al (1951)
using bovine serum albumin as a stand ard and reading the absorbances on a
sp e c tro p h o to m e te r (Spectroplus 20) at 660 nm.
10. Production of specific antibodies
For t h i s purpose th e method su gg ested by Harboe and Ingild (1973) was used. The
p u rifie d antigen p ro te in was diluted in saline to a concentration of 4mg/ml and
3-4 kg ra b b its w ere used as experimental animals in which the antibodies were
r a i s e d . On days 0, 14, 28 and 42 each rab b it was given a stan dard mixture
co n sistin g of 50/ jI antigen solution ♦ 5 ^ 1 F reu nd 's incomplete adjuvant. This
mixture was injected very superficially subcutaneously in the thicker p a r t of
th e skin over th e scapula. Or
,
sample of up to 45ml of blood was
w ithdraw n from a la t e r a l ear vein.
an a lte rn a tiv e up to 60ml of blood was
ob tain ed by cardiac puncture in -
- t i s e d r a b b i t s . Blood sampling was
r e p e a t e d every six weeks, pr.
anim als had received a b o oste r injection
of th e stan d ard mixture 8-10 days before.
A f t e r c e n trifu g atio n the plasma sam ples were frozen and sto re d . Plasma obtained
from each ra b b it on s e p a r a te d e e d in g occasions w as pooled for iso latio n of the
IgE and IgA.
To each 100ml antiserum 25g ammoniumsulphate w as added and the mix. . .
as
allowed to s t a n d for about 20 hours at room tem p e ratu re. A fter ce ntrifug atio n
for 30 minutes a t 4,000 xg 98% of the antibody was precipitated and the
s u p e r n a ta n t could be discarded.
T h is p re c i p it a te w as th en washed with about 25ml 1,75M ammoniumsulphate
s o lu tio n , centrifuged and the su p e r n a ta n t again d iscard ed. This washing was
r e p e a t e d once more. A fter dissolving the p rec ip itate in a small amount of w ater
the mixture w as placed in a 25ml d ia lysis bag and dialysed at 4% for two
12-hour period s a g a in s t d is tilled w ater, one 24-hour period again st an acetate
b u f f e r <0,05M N a -a c e ta te + 0.021M H -a c e ta te , pH = 5), two 12-hour periods
a g a in s t d is tille d w ater, and again one 24-hour period a g a in s t the acetate
b u ff e r. The sample was then again centrifuged a t 4,000 xg for 30 minutes and
the p re c ip ita te (lipoproteins) discarded.
The s u p e r n a ta n t w as th en t r a s n s f e r r e d to a column containing 25ml CEAK Sephadex A50 equ ilibrated with the a c e ta te buffer (pH = 5). The column was
elu ted with about 25ml a c e ta te bu ffer. The eluate w as th en concentrated by a
s a l ti n g out p ro c e s s using 25g ammoniumsulphate per 100ml eluate anu the,
dialy sed ag a in s t 0,1 M NaCl for tw elve hours a t 4°C. The final solution was
frozen and s to r e d a t -20°C.
results
C ellulose a c e ta te ele c tro p h o re sis was used to follow the success of ti
two
albumin iso la tio n methods and Lowry g i l l ' s to t a l p ro te in estim ation
method was used to calculate the percentage pro tein yield. The polyethylene
gl,
; " i ii c it d ti o n method yielded above 80% of the available albumin but
s u b s t a n t i a l amounts of im purities were present.
The eth ano l fra c tio n a tio n method, on the other hand, yielded co nsistently more
th an 90% of the av ailab le albumin and contained no electrophonetically
d e te c ta b le im pu rities. For th ese re as o n s, and also because of the ease of
methodology, th is method was p re fe rred and used fo r the isolation of the
ex pe rim e nta l sam ples. An example of the cellulose electrophoretic sep aratio n of
lion plasma in the d if f e r e n t s t a f e s of isolation compared to th a t of a human
sample can be seen in Fig 1. In both in stances "pure" albumin in the
c h a ra c t e ri s ti c configuration was obtained with only trace amounts of unwanted
se p a r a ti o n products.
EXPERIMENTAL MANIPULATION
In all th e following experim ents lion whole plasma a s well as the isolated
lion plasma albumin w ere used in all the d ifferen t techniques.
A. C ellulose a c e ta t e elec tro p h o resis
This s e p a r a tio n method was used in the in v estig atio n of the following:
/
Y-
34 .
OTHER HUMAN PLASMA
PROTEINS( PRECIPITATE)
ISOLATED HUMAN ALBUMIN
(SUPERNATANT)
HUMAN PLASMA
ISOLATED LION ALBUMIN
(SUPERNATANT)
LION PLASMA
FIGURE 1 . QUALITY CONTROL OF THE ISOLATION PROCEDURE.
1 E sta b lis h in g the ch ara c te ristic p a tt e r n
Plasm a sam ples r r e ig h t lions were initially rep eate dly s e p a r a te d and all
s e p a r a ti o n s showed the same p a t t e r n of a high concentration f a s t running albumin
band followed by a low concentration slower running band (Fig.2).
2 C om oanson with human plasma albumin
All e ig h t lion plasma samples were compared to human plasma sep arated on the
same s t r i p s and under identical conditions. This comparison highlighted the
d i f f e r e n t p a t t e r n s of albumin with the single human albumin band and the double
lion albumin bands. It also revealed the c o n s is te n t p a tte rn of f a s t e r running
lion albumin bands (Fig.3).
3 Comparisons with o ther species
S e p a ra tio n s of plasma from cheetah and oomestic cat were performed on the same
s t r i p s as lion and human plasma. All the cat sp ecie s had f a s t e r running
albumins compared to the human (Fig.4).
4 Comparisons betw een lions of d if fe re n t a re a s
The sample population was extended to include lions from all O' er Southern
A frica including th e E a s te r n T ransvaal (Kruger National Park), Northern
T ran sv aal, Kalahari D esert and Northern Namibia (Etosha Game Reserve). This
made up a sample size of 65 lions and th eir electrophoretic p a t t e r n s were all
ide ntic al.
5 Influence of time from immobilization to sampling
Previou s workers (Melton e t al, 1987) reported an e ff e c t of th i s t ’ns
in te rv a l on the to t a l plasma p ro tein composition. The p re s e n t in v estigatio n
found no d if fe re n c e s in the sep ara tio n p a tt e r n s re g a r d le s s of th e elapsed time
(up to 1,5 hours).
6 Influence of immobilization drugs
The two drugs used in immobilizing th es e animals were xylazlne hydrochloride
SEPARATION PATTERN OF LION WHOLE PLASMA.
SEPARATION PATTERN OF HUMAN BISA LBUM INEM IC PLASMA
FIG URE 2 . CELLULOSE ACETATE ELECTOPHORF.TIC SE P aR A P IO N .
FIG U R E 2 b
DENSITOM FTRIC A N A L Y SIS OF ELECTOPHORETOGRAM
LION ALBUMIN
HUMAN ALBUMIN
LION ALBUMIN
HUMAN ALBUMIN
HUMAN
CHEETAH
DOMESTIC CAT
FIG URE U.
COMPARISON AMONG VARIO US D IFFE R E N T SP E C IE S
AFTER C E l JLOSE ACETATE EL EC TR O PH O R ESIS.
(Rompun; Bayer, West Germany) in combination with ketamine h yd roth.onde
(V etalar; P ark-Davis. USA) and phencyclidine hydrochloride (Sernylan;
B io-C eutic L a b o ra to rie s Inc, USA). The e f f e c ts of a n a e sth e tic drugs on other
blood chemistry and haematological p aram eters nave been reported (Steyn, 1975).
Although the p ro te in content of plasma could be affec ted , th is study showed no
d ifferen ce in p ro te in se p a ra tio n s.
7 In f lu e nce of antl-co aq u lan t
In t h i s in v e s tig a tio n blood was collected in e ith er heparin, oxalate or citra te
b e fo re cen trifug ation ar altern a tiv ely allowed to clot before centrifugation.
The se p a r a tio n of the d if fe re n t plasmas as well as serum were as fa-- as the
albumin p a t t e r n was concerned, all similar.
8 The e ff e c t of storing
I t i s well known th a t sto rin g plasma at -4 C in a fre eze r does not -ompletely
p re v e n t sam ples undergoing changes asso ciate d with protein d enaturation,
possibly due to continued enzymatic activ ity . In addition to th is , frequent
thawing and refree zing may be p a rt of the usual labo rato ry procedure where
p o rtio n s of sam p les are used and the remainder sto re d again. The e ff e c ts of
such in t e r m it te n t freezing and thawing h a v been described for other wild animal
sp e c ie s (Cheney, 1982) and in th is inve stiga tion the same general changes as far
a s t o t a l p ro te in and albumin concentrations could be observed in lion plasma.
The ele c tro ph oretic p a tt e r n , however, was not a lte re d . Frequent thawing and
refreezing w as avoided by dividing all samples in to small aliquots which were
s t o re d , used once and discarded. Total pro tein and albumin concentrations could
th e r e fo re only be affe cted by minimal changes occuring during long term storag e
a t -4°C.
9 Effec* of length of se p a ra tio n time
A possible methodological explanation for a c e rta .n elec trophoretic sep aratio n
configuration could be the time allowed for se p a ra tio n which would ‘hen
influence the d istance of movement of various fra ctio n s. Short d istan ce s could
th u s r e s u l t in inadequate se p a ra tio n with one fraction obscuring o th e rs and
longer dis* nces may allow for sp littin g sin g le -ty p e p ro te in s into subunits,
making comparisons to stand ard p a t t e r n s very diffic ult. Ele ctrophoretic time
was v a rie d from 10 minutes to 45 minutes. In all se p a ra tio n s la s tin g a s h o r te r
time th a n the sta n d ard 20 minutes an inadequate sep aratio n could be observed
th a t n o t only obscured the second slower albumin band but als o o ther plasma
p ro te in fra c tio n s (Fig.5). All the s e p a r a tio n s over a longer time revealed the
two albumin bands but spread the bands of o th e r fraction s so wide th a t they
became very ind istin ct and difficult to id entify.
10 E f f e c t of d if fe re n t concentrations of sample
Since loading of d if f e r e n t amounts and d iffe re n c e s in concentration of samples
can in fluence the clarity of electrophoretogram s and change the behaviour of
p ro t e in s (especially in very dilute concentrations) a comparison was made
b etw ee n th e se p a r a tio n of dilute sam ples. Plasma was diluted with isosmotic
saline and Fig.6 show s the se paratio n o f a l + 2 and a 2 + 2 dilution compared
to undiluted plasma. In th e s e two dilutions the pro te in s of sm aller
con c en tra tio n become progressively fa in te r but th e double-banded albumin is
maintained. F u rth e r d ilution s ( 1 + 3 or 1 + 4 ) yielded a very fa i n t albumin
p icture th a t , however, s t i l l retained the same configuration. More concentrated
sam ples (concentrated once and five-fold , respe ctiv ely , in a Minicon
microconcentrator) a s well as increased amounts of sample loaded gave a very
obscure ele c tro ph oretic p a tte rn , to tally impossible to identify.
11 Comparison betw een sample s from wild and captive animals
R eported values of the plasma to ta l p rotein and albumin concen+rations for wild
lions (Melton e t a l , 1937) d iffer slightly from th o s e of captive lions
(Morse and Follis, 1974 and Fowler, 1973). In th is study a small sample of
e ig h t lio ns were in fa c t from a captive population which may, mainly from the
1 0 MINUTES
2 0 M INUTES
4 5 MINUTES
FIG U R E 5 .
THE EFFECT OF SEPARATION TIME ON CELLULOSE
ACETATE E L EC TR O PH O R ESIS.
43
1 + 2 D IL U TIO N
2 + 2 DILUTION-
UNDILUTED
FIG URE 6 .
THE EFFECT OF SAMPLE D IL U T IO N ON SUBSEQUENT
CELLULOSE ACETATE EL EC TR O PH O R ESIS.
po int of view of a d if f e r e n t d iet, have had a slightly modified plasma
composition. Comparison of the e lec trophoretic p a t t e r n s , however, did not yield
any noticable d if fe re n c e s .
12 E f f e c t of feeding
K tending the in v e s tig a tio n of the influence of diet, a sm all group of eight
captive lien s were used to observe p o ssib le changes caused by the qu an tity of
food given a t feeding se s s io n s . These lions w?"e s t a r v e d for two d ays, given
norm al r a t io n s
r two days and th en double ra tio n s fo r two days with a suitable
eq u ilib ra tio n time in betw een each se s s io n . Figure 7 show s the comparison
b etw e en the electrophoretically se p a r a te d samples of th e plasma obtained a ft e r
each of th e s e th r e e s e s s io n s . It is obvious th a t qu an tity of food e a t e n does
n ot a l t e r the p ro te in p a tte rn .
13 P ercentage composition of the albumins
From th e d e n sitro m e tric scanning of the cellulose a c e t a t e s trip s (fig 2b) and
the to t a l p ro te in e s tim a tio n s the following overall r e s u l t s for the whole
population were obtained (MeantS.D., n = 65)
Mean to t a l p ro te in concentration in plasma = 8 3 ,8 1 3 ,3 g /l
Mean albumin c oncentration in serum and plasma = 37,3 t 7,2g/l
Mean c oncentration of anodal albumin band » 30,7 t 5,98 g/1
Mean c oncentration of cathodal albumin band = 6 ,5 4 1 3 ,2 2 g/1
B. Pglyasryljm .d? d ^ f q el.electrpp h o resis
In t h i s se p a r a tio n technique it is possible to a lte r the gel concentration a
hence th e pore size of the gel. Initial sep a ra tio n of lion plasma v as done
rep eate d ly on a range
of gel concentration media: 3%, 5%, 7,5%, 8,5%
and 10%.
The same double banded p a tt e rn was observed and it proved not to be a function
of medium den sity (Fig.8ai. The 7,5% gel yielded the c l e a r e s t p a tt e rn and was
a ) OVERFED
FIGURE 7
THE EFFECT OF FEEDING STATUS ON ELECTROPHORETIC
SE PARATION PATTERN .
b) NORMALLY Ffib
c)
STARVED
FIGURE 8a THE EFFECT OF DIFFERENT GEL CONCENTRATIONS ON
DISC GEL ELECTROPHORESIS.
—
------------
U
t h e r e f o r e chosen fo r su bseq ue nt s e p a r a tio n s , also including the s e p a ra tio n of
the p urified albumin (Fig.Sb)
This method w as used to in v e s tig a te some of the same influences te s te d on
microzone cellulose s trip s :
1 E s ta b lis h in g th e in itia l p a tt e rn in eigh t lion plasma samples
2 Comparison with human plasma
3 Comparisons betw een 65 lions from d if fe re n t geographical a re a s
4 Influence of length of period; immobilization to sampling
6 Influence of d if f e r e n t a n ti-c o a g u la n ts
7 E f f e c t of sto rin g of sam ples
8 E f f e c t of length of sep aratio n time
9 Po ssible d iffere n c es betw een wild and captive sample animals
10 Influence of amount of sample. Q u an titie s of 1, 2 or 3/ j 1 were compared.
In all th e s e in v e s tig a t. ns the sep aratio n p a t t e r n s were similar.
C.
A r . i y t j r a l . s o e l e c tn c focusing
This method s e p a r a t e s pro te in s into very fine fr a c tio n s which makes
id en tific a tio n d ifficult since stan dard p a tt e r n s are fairly irregu lar in
configuration. In th is study, on sepa rating lion plasma, i t was possible to
id entify th e "albumin zone" by comparing it to the is o elec tric po in ts of human
albumin and o th er marker p rotein s, eg haemoglobin. On th is b a s i s i t is possible
to s t a t e th a t the albumin zone for lion albumin is qu ite distinctly d iffere n t
from t h a t of human albumin and fu rth e r th a t th is zone is similar in all lion
sam p les (Fig.?). The possible differenc es in focusing p a tt e rn was in vestig ated
for th e following influences:
FIG U R E 8 b .
SEPARATION OF LION ALBUMIN BY POLYACRYLAMIDE
D IS C GEL E L EC TR O PH O R ESIS.
I.ION PLASMA
HAEMOGLOBIN
HUMAN
ALBUMIN
MARKER
FIG URE 9 .
ALBUMIN ZONES OBTAINED BY ISO E L E C T R IC FOCUSING
A
t-
--------------- -----
■Mi
1 Comparison? b etw e en 65 lio n s from d if fe re n t geographical *reaS
2 Ccmpari'-
mman albumin
j In fluenct
i of period: immobilization to sampling
5 Influence of d if f e r e n t a n ti-c o a g u la n ts
6 E f f e c t of sto rin g of samples
7 D ifferen ces b etw een wild and captive animals
8 Influence of length of focusing period
It i s Known th a t an increase in the duration of focusing will r e s u lt in fatty
acids being removed from albumin, so th a t the aibumu. will <n fact undergo
d e f a t ti n g (Basic £ i al, 1978). Sho, .d the focusing p a t t e r n be
influenced by t h i s combination of fa tty acids and albumin, t h i s s e p a ra tio n of
th e tw o s u b s ta n c e s would a l t e r the focusing p a tt e r n . Lengthening the focusing
time to th re e ho urs had no influence and Fig.10 shows a typical iso electric
focusing p la te.
D.
r.n lu m n e x c l u s i o n c h r o m a t o g r a p h y
Recordings made of xhe lion plasma sample-containing b u ff e r showed a very clear
is o la t io n of the albumin fraction a s a whole but s e p a ra tio n of the two
components could not be achieved. Reducing the flow to 2ml/hour s t i l l did not
r e s u l t in producing any s p lit in the albumin peak. Is o la tio n of the two albumin
fr a c ti o n s could th u s not be achieved by column chromatographic methods, in spite
of using su perfine Sephadex 200 in a very large volume column at an extremely
slow flow velocity (0.2 ml per hour) .
E.
Prepa ra tive IEF
By employing a blottin g technique using a s h eet of f i l t e r paper, the isoelectric
FIG URE 1 0 .
ISO E L E C T R IC FOCUSING OF LION P1ASMA
focusing p a tt e r n could be determined. From th is i t became clear th a t sep aratio n
of t h e two albumin t y p e s would be difficult, they focused very close to g e th e r
and in a wavy f r o n t . Cellulose a c e t a te e lec tro p h o resis of the resuspended
" i s o la te d " sam p les eluted from the medium showed the tw o-band p a tte rn appearing
in b o th fr a c tio n s cut out of the pre parativ e gel.
F. SDS disc oel e le c tro p h o r e s is
In t h i s method th e d ete r g e n t covers the charged s i t e s on the partially denatured
p r o t e i n s so t h a t se p a r a tio n occurs more on the b a s i s of molecular mass than
c h a rg e . In t h i s in s ta n c e all the modifications of t h i s technique (varying gel
c o n c en tra tio n by changing the amount of concentrated acrylamide bisacrylamide
added and also varying the amount of sample added) s t i l l proouced only a single
albumin band (Fig. 11).
In t h i s r ethod v a rio u s a tte m p ts were made to modify the procedure to e li m i n a t e
any influence p o ssib ly caused by the procedure. For example, samples were
e i t h e r stained o r n o t stain ed with the tracKing dye (bromophenol blue) and were
e i t h e r subjected or not to h e a t d enatu ratio n. Only th ose suojected to both
t raCKing dye p re s ta in in g and h e a t denaturation yieloed a s a t i s 'a c t o r y sep aratio n
p a t t e r n . These sam ples also divided into only one albumin band.
H. C rossed im m uno-electroohoresis
T h is was done by using agarose slab gels, polyacrylamide disc gels, iso elec tric
focusing and SDS polyacrylamide disc gels sep aratin g both lion plasma and lion
albumin into t h e i r respective su bfractio ns in the f i r s t dimension. Th» second
dim ension c o n s is te d of agarose containing e ith e r anti-hum an albumin (Dako) or
a n t i - l io n albumin (obtained from rab b its in our laboratory) antibodies. The
55.
FIG URE 1 1 .
SDS -
POLYACRYLAMIDE D I S C GEL ELECTROPHORESIS
s e p a r a ti o n on the agarose was not d is tin c t and revea.ed th e true two-banded
album in p a t t e r n only when the application of the sample w as done in thin strip
form in s te a d of the usual well. SDS seemed to in t e rfe re with the antibody
binding re a c tio n in the seconc dimension and th u s the method of using SDS
polyacrylamide disc gels was discardeo.
F ig u r e s 12 and 13 show the p a tt e rn obtained by polyacrylamide disc gels in the
f i r s t dimension sep aratin g human albumin and lion albumin respectively and using
a nti-hu m a n albumin antibody in the second dimension.
F ig u r e s 14 and 15 show the p a tt e r n obtained from f i r s t dimension re p a r a tio n in
a g a ro s e g e ls of human and lion albumin respectively and t h . r reacting th a t
a g a in s t anti-hum an albumin antibody.
F ig u re s 16 and 17 show the p a tt e rn obtained from f i r s t dimension sp littin g of
human and lion albumin respectively on IEF and then obtaining the reactio n with
anti-hu m an albumin antibody.
F ig u r e s 13 and 19 show the p a tt e rn obtained from in i ti a l sep ara tion of human and
lion albumin respectiv ely on IEF and su bseq uen t r e a c t io n s with a n ti-lio n albumin
antibody.
In all of th e s e , despite sm aller d iffere nce s due to varying amounts of
background p ro te in s not taking place in the specific re a c tio n and also because
of th e rela tiv e ly f- o u re n ature of the non-commercially manufactured an ti-lio n
albumin antibo die s, the configuration of the crossed re a c tio n was sim u a r.
Human albumin s e p ara ted into one d is tin c t band th a t re a c te d in one smooth and
reg u lar peak with both anti-human and a n ti-lio n an tib o d ies . Lion albumin
F IG U R E 1 2 .
CROSSED IMMUNOELECTROPHORESIS OF HUMAN ALBUMIN
A G A IN ST ANTI-HUMAN ALBUMIN A N T I B O D Y .
F I R S T D I M E N S I O N : AGAROSE EL EC T R O PH O R E SIS.
SECOND D IM EN SIO N : AGAROSE EL EC TRO PH OR ESIS.
F IG U R E 1 3 .
CROSSED IMMUNOELECTROPHORESIS OF LION ALBUMIN
A G A IN ST ANTI-HUMAN ALBUMIN AN TIBO D Y .
F I R S T D IM E N SIO N : AGAROSE ELECTRO PH ORESIS.
SECOND D I M E N S I O N : AGAROSE EL EC T R O PH O R E SIS.
/*
FIG URE 1 4 .
FIG URE 1 5 .
A G AIN ST ANTI-HUMAN ALBUMIN A N T IB O D Y .
F I R S T D IM E N S IO N : D I S C GEL ELECTROPHORESIS
SECOND D IM E N S IO N : AGAROSE EL EC TRO PH O R ESIS.
CROSSED IMMUNOELECTROPHORESIS OF LION ALBUMIN A G A IN ST
AG AIN ST ANTI-HUMAN ALBUMIN AN T IB O D Y .
F I R S T D IM E N SIO N : D I S C GEL EL EC TR O PH O R ESIS.
SECOND D IM E N SIO N : AGAROSE EL EC TRO PH O R ESIS.
FIGURE 16
AGAINST ANTI-HUMAN ALBUMIN ANTIBODY.
FIRST DIMENSION: ISOELECTRIC FOCUSING.
SECOND DIMENSION: AGAROSE ELECTROPHOR!
FIGURE
.
AGAINST ANTI-HUMAN ALBUMIN ANTIBODY.
FIRST DIMENSION: ISOELECTRIC FOCUSING .
SECOND DIMENSION: AGAROSE ELECTROPHORESIS.
FIGURE 1 8 .
A G A IN ST A N T I - L I O N ALBUMIN AN T IB O D Y .
F I R f . D I M E N S I O N : AGAROSE E L E C T R O PH O R E SIS.
SECOND D IM E N SIO N : AGAROSE EL EC TRO PH O R ESIS.
FIGURE 1 9 .
A G AIN ST A N T I - L I O N ALBUMIN A N T IB O D Y .
F I R S T D IM E N SIO N : AGAROSE EL EC TRO PH O R ESIS.
SECOND D IM E N SIO N : AGAROSE EL EC TRO PH OR ESIS.
s e p a r a te d in to the ch a ra c te ristic double-banded p a t t e r n which th en reacted in
one smooth and continuous peak with a "toe" indicating the reaction of the
sm a ller more cathodal albumin band. This in dicates th e expected
c r o s s - r e a c t i v i t y betw een albumin and specific an tibo dies of d if fe re n t species
(human and Hon). More im portantly, i t re v e als th a t the two lion albumin bands
are immunologically s i m i la r.
62.
DISCUSSION AND CONCLUSIONS
From the r e s u l t s of th e s e in v e s tig a tio n s i t is clear th a t th e is olation
p ro ce d u res r e s u l t e d in a reasonably purified albumin. Pure albumin in th is
c o n te x t would mean a s u b s 'm c e not accompanied by o th e r '.peptide or non-peptide)
s u b s ta n c e s . I t m ust be re a lia e c , however, th a t th is scavenger-like p ro te in would
inv ariably be carrying ligands brvnd to it. Pu rity , th u s , would he basec on
c o h e re n t g en eral behaviour like precip itation c h . r a c t e r i s t i c s , migration ra te
through gel columns a.id size-an d cnarge-based elec trop ho retic sep aratio n
p a t t e r n s bu t would acknowledge the ex istence of bound p a rtic le s not originally
s y n th e siz e d w ith the albumin. This was already noticed decades ago (Kendall,
194D
when even rep eate dly crystallized human albumin did not give solubility
c u rv e s c h a ra c t e r i s ti c of a pure substance. This is also th e underlying cause
for is o e le c tric focusing showing an albumin region of d issim ilar bands r a t h e r
th a n a single albumin fractio n. The existence of th ese multiple albumin forms
t h a t d if fe r slig htly in charge or solubility is re f e rre d t : as the
m icro hetero gen eity of albumin. Although the fa c to rs involved in causing th ese
changes are numerous and varied , they have in common th e fact th a t they all
occur a f t e r s y n t h e s i s of th e molecule and th us cause p o s t - t r a n s la t io n a l
modifications to albumin as it circulates through the body.
The following dis cussio n of albumin genetics, s y n th e sis , amino acid sequence,
isomeric forms and the concept of microheterogrneity is based on the review
a r t i c l e s a vailab le th a t deal with th ese overall principles on a general b a s i s
( F o s t e r , 1977 and P e t e r s , 1985).
63.
MICROHETEROGEKE-ITY OF ALBUMIN
F a tt y acids are bound tig htly enough to remain with the albumin during
e l e c tro p h o r e s is or ion-exchange chromatography. At the pH conditions of
is o e le c tric focusing they a re slowly removed from the albumin. Human d e fa tte d
albumin will focus a t pi 5.6 and fa t ty - a c id containing albumin a t pi 4.3. By
lengthening the focusing period the former band would increase in in ten sity
while the l a t t e r would decrease. This migration of f a t ty acids to w ard s the
anode most probably did occur with the lion samples but did not seem to a lt e r
th e o verall p a t t e r n of s e p a ra tio n . These fa tty acid molecules (mostly in th e ir
so a p or s a l t form) bind with decreasing affin ity to the albumin, usually one or
two pe- albumin molecule, rising to no t more than four in high t i t r e fa tty acid
s i t u a t i o n s (e g .a fte r exercise). The binding s i t e s for th e s e ligands on human
albumin seem to be the middle of domain III, the border between domains II and
III, middle of domain I and the middle of domain II for the f i r s t four ta t ty
acid molecules re s p e c tiv e ly . The binding seems to occur ir. two d i s ti n t
ste p s:
f i r s t l y a rapid but lo ose attachment to the albumin molecular su rface f n lo w e d
secondly by an opening of a hydrophobic pocket to allow the entry of the
hydrophobic ligand p ortio n into the in te rio r of the molecule. This res u ts in
th e formation of an ionic bond Between the carboxyl of the fa t ty acid ano a
cationic amino acid sid e chain. This binding, of especially the f i r s t two fa t ty
acid molecules, changes the shape of the fatty acid molecule from an elongated
oval to a stubby and r .under shape which one would expect to be the prevalent
molecular form in plasma.
Bilirubin can also r e s u l t in an anodic exten sion of the albumin b.n d on agarose
e lec trop ho re tog ram s. There appears to be only one strong binding s ite for
64,
b ilirub in which is in domain II. Binding here r e s u l t s in unfolding of the
bilirub in molecule by breaking the hydrogen bonds and th en holding it in a
ti g h t, highly tw is te d o rien tatio n in a locus which is p rotected from the
surrounding medium. This binding occurs as an alm ost in s ta n tan eo u s assoc iatio n
of bilirubin with albumin, followed by a s e r ie s of relax atio n pro c esses to reach
th e final configuration. The binding of the bilirubin is affected by the number
of f a t t y acids bound to the albumin where large numbers of fa tty acid molecules
will compete with and de p ress the bilirubin binding w hereas sm aller numbers of
f a t t y acids will actually enhance bilirubin binding due to a decreased
a lt e r a t io n in sh ape of the aloumin molecule. Albumin from the umbilical cord
and in prolonged uremia also shows a decreased capacity (with normal affinity)
fo r binding bilirubin due to an unknown ligand in each in stance partially
blocking the biliru bin s i t e . Charcoal tr eatm e n t a t pH 3 c lea rs the aloumin of
t h e s e ligands and r e s t o r e s the bilirubin binding capacity.
The single thiol group (on Cys.34) in domain I is a r e s tr i c te d hydrophobic s ite
t h a t can bind c y stin e and glutathione for tr a n s p o r t from the gut to the
p erip h e ral t i s s u e s . This binding can cause h e te r o g e n e it y at th a t s ite t h a t can
influence the p a t t e r n s obtained a ft e r ion exchange chromatography.
Albumin is a non-glycoprotein, on of the few se c re te d p ro tein s to lack
carbohydrate, bu t nonenzymatic glucosylation i n s e r t s a monosachande onto about
3% of albumin in the normal individual (and considerably more in the
uncontrolled d iab etic with prolonged hyperglycaemia). The binding s ite for th is
monosacharide is in domain III.
T reatm en t of rheumatic d is e a s e s very often involves prolonged exposure to acetyl
salicylic acid which can cause acetylation of albumin in domain II. Similarly,
#
1
mostly tr a n s ie n t , h etero g e n e ity can be observed by e le c tro p h o re sis because oi
t r e a t m e n t with c e rt a in penicillins, ethacrynic acid or mercaptopurine.
Circulating albumin will also exhibit an ever increasing degree of molecular
s t r u c t u r a l changes. Isom eration by disulfide in terchanges occurs with
increasing age and th i s can a ff e c t up to 10% of circulating albumin.
T ra n s ie n t abn orm alities can be observed in p a ti e n ts with pancreatic cy sts where
p ro te o ly tic enzymes a l t e r the C-terminal of the normal albumin to create a
fas te r-m o v in g band on ele c trop ho re sis.
Gradual modifications also occur to side chains of amino acids, like in all
circulating p ro te in s , and deamination of asparagine a d glutamine has been
implicated in the cause of m icroheterogeneity. Nonenz, matic conversion of
methionine r e s id u e s to methionine sulfoxide also occurs in the circulating
p r o t e in s of c e rta in individuals.
T hese ligands, e it h e r bound tightly but reversibly or bound covalently to
albumin or th e s e s tr u c tu r a l changes of the albumin molecule can th u s render the
albumin very subtly heterogeneous and can influence c e rta in analytical
procedures. Only th o s e not involving ad m inistration of specific drugs and those
n ot resu lting from a specific d isea se (like d ia b e te s of pancreatic cysts) could
possibly a ff e c t the e n tire lion population as described. For an explanation of
th e lion p a t t e r n a t t e n ti o n should then s h i f t to changes imparted to the albumin
se p a r a tio n by a c o n s is te n t in terfe re nce from the binding of naturally occuring
lig an ds in +he circulation. This should th en happen to such an e x te n t th a t the
albumin p a t t e r n will be affected not only in highly refined analytical sy stem s
like iso electric focusing but also co nsisten tly in comparatively crude
s e p a r a ti o n a n a ly s is like cellulose a c e ta te ele c tro p h o re sis or polyacrylamide gel
e le c tro p h o r e s is . Ligand binding or molecule modification having such a
pronounced e ff e c t i s v irtually impossible and h as no t been rep o rted in any
p rev io us work. F urtherm ore, the numerous d if fe re n t analytical methods and th eir
many modifications should have illuminated the influence of any of th ese
abovementioned f a c t o rs in the sen s e of showing some changes in the sepa ration
con fig uratio n due to the differen t methods imposing diffe ren ces in ligand
binding c h a r a c t e r i s ti c s on the albumin molecules. The few differen ces in the
sample population (eg feeding p a tte rn , age, sex) should also change some of
t h e s e ligand a tta c h m e n ts which should influence the eventual se p a ra tio n . The
fact t h a t th e same p a t t e r n of se p a ra tio n was observed in sp ite of the d iffere n t
donor s o u rce s, degree of refinem ent of s e p a ra tio n technique and sample
manipulations po in ts to more significant a l t e r a t io n s to the albumin of lions
than th o s e imposed by sligh t molecular modifications or ligand binding
influen ces.
SYNTHESIS OF ALBUMIN *ND ITS AMINO ACID SEQUENCE
A more likely source of v a r i v r n lie s in the s y n t h e s is p ro c e s se s of albumin.
These obviously have a genetic base or r e s u lt from a lte r a t io n s of the normal
p ro c e s s of albumin sy n th e s is . Albumin RNA has been is o late d from liver cell
cytoplasm and from th e s e , by cloning the complimentary DNA in b acteria, the
nucleotide base sequence of the albumin gene h as been e sta b lish e d for a number
of sp e c ie s. The p o sitio n of the gene on the chromosome has been found to be in
th e same region as t h a t of alpha feto p ro te in , the embryonic counterpart of the
adu lt albumin which is transcribed from the same stran d of DNA double helix.
T hese genes are about ten times as long as the coding segment and in both
albumin and alpha feto p ro te in coding segments th ere are fou rteen in tron s,
c re a tin g f i f te e n exons. The le ng ths of th es e exons form a p a tt e r n of t r ip l e t
homology t h a t correspo nd s to the three albumin domains while the s i t e s of
i n s e r t i o n of the in tro n s correspond to regular loci in the albumin chain. One
f i n d s t h a t the in te rn a l th re efo ld sim ilarity p a tt e r n is much more homologous in
th e DNA sequence th an in the pro tein amino acid chain, stren gth enin g the concept
t h a t th e p r e s e n t day albumin aro s e ■
‘rom a single domain or even ju st a single
loop, the gen es fo r which underwent replication.
Albumin s y n t h e s i s in the hepatocyte follows the p a tt e r n of all se c re te d proteins
when 19 ribosom es attach to one albumin mRNA to form a large polysome. This
complex is guided to the membrane of the endoplasmic reticulum by a so called
sig n al p eptid e, which is th e f i r s t tr a n s la te d sequence and which shows a
rem arkable i n t e r - s p e c ie s homology. This signal peptide d ire c ts the growing
p ep tid e chain through the membrane of the -ough endoplasmic reticulum and is
th e n cleaved o ff , even b efore the tr a n s la tio n of the mRNA is completed.
Such newly formed albumin, s t i l l in the secreto ry channels of the liv er cells,
i s initially in th e form of proalbumin which is normal albumin with a
he xapeptide attac h e d at i t s amino terminus. In th is form the protein migrates
from rough to smooth endoplasmic reticulum and to the Golgi complex where the
le a d e r peptide i s cleaved o ff ju s t betore re le a s e from the cell. Proalbumin is
no t normally found in the circulation and only mutations of the hexapeptide
sequence th a t cause a blocking of the cleavage will r e s u l t in th is leader
p eptid e being retain ed on the secreted molecule. Cleavage then ta kes place in
the circulation, seemingly with the liver as the responsible organ.
This th en r e s u l t s in the single chain molecule of which the peptide sequential
composition i s known in a few species. The existence and alignment of 17
I
41
disulphide bridg es c re a te the b a s i s for the unique albumin molecular
con fig uratio n of a s e r i e s of 9 loops repeated in a t r i p l e t fashion of
la r g e - s m a ll - la r g e loops making 3 homologous domains of 3 loops each. The amino
acids are not d is tri b u te d evenly; some favour th e amino half of the chain and
o t h e r s the carboxyl h alf. The molecule is also not uniformly charged along i t s
length with the amino head highly negative and th e carboxyl ta i l l e s s negative
or n e u tr a l. Loop homology in dicates th a t the large molecule could h a-e evolved
from a precurso r of o n e-th ird or even one-ninth the p re s e n t size, as was
mentioned above.
I n t e rs p e c i e s sim ilarity is quite high betw een bovine and human and betw een r a t
and human (about 30% each) but slightly lower (about 63%) among all 3 species.
Sim ilarity betw een a lp h a - fe to p ro te in and albumin is a lot lower (less than 40%)
in both humans and r a t s , with the homology more in the carboxyl end p a rt of the
molecules. A lp h a-feto p ro te in h as no propeptide s t a g e during i t s b io s y n th esis
and h a s, a p a r t from about 4% carbohydrate, a much more selectiv e affinity for
ligands th an albumin.
The study of the e f f e c t s of breaking peptide bonds without breaking disulphide
bonds revealed th a t a v ariety of fragments can be obtained in t h i s way.
Iso la tio n of th e s e p e p tid es has confirmed the disulphide-bonded loop s tru c tu re ,
h as helped to localise binding functions, has de m onstrated the autonomy of
t e r t i a r y stru c tu re of certain p a r t s of the molecule and has helped to localise
antigenic d e te rm in an ts. Loop 9 contains no ty ro sin e or tryptophan but contains
antigenic s i t e s to tne e x ten t th a t iso lated bovine loop 9 fragm ents can prime
anim als for a secondary immune resp on se to whole albumin or, when injected in
the developing immune system , can give tolerance to the whole albumin molecule.
In human loop 9 the active region h as been determined as amino acids 545-373 and
monoclonal a n ti b o d ie s to the region have been manufactured.
P re c ise knowledge of the exact amino acid sequence has enabled r e s e a rc h e rs to
calc u la te th e accurate molecular mass and, in species where th is has been
p o s s ib le , t h e s e calc ulated m as ses are remarkably c o n s is te n t with e stim a tio n s
made p reviously on a hydrodynamic b asis. Likewise, the nitrogen content of
albumin can th e n be calculated more accurately and slig h t modifications can be
made to the assum ed 16% conventionally adhered to. For lion albumin th e s e
v a lu e s , h is to ric o r a ltered by specific molecular mass calc ulations, are not
known.
Evidence o btain e d from sedim entation s tu d ie s , die lectric dispersion, electriu
birefrin g e n ce, low angle X-ray s catte rin g and electro n microscopy confirmed the
ou tlin e shape of the albumin molecule as being ellipsoid, not to tally filled by
polypeptide b u t having a hydration shell of w ater molecules, some a t specific
fixed loci and o t h e r s more widely d is trib u te d but s t il l tig htly enough bonded to
th e protein by hydrogen bonds to render th is w ater n o n-free zab le. In n atural
albumin th e r e i s most probably 1 - 2 w ater molecules per amino acid residu e.
The r e f ra c tiv e index increment of albumin is very typical of a simple protein,
co nsistin g a lm o st only of polypeptide chains. U ltrav io let light can be used to
t e s t the absorbance of amino acid aromatic side chains or peptide bonds,
depending on how the protein is prepared before spectrophotom etry. Fluorescence
o f albumin i s determined by i t s tryptophan re s id u e s and the ro tatio n of
polarized lig ht by an albumin solution is typical of a globular protein.
From th is b r i e f description of albumin s y n t h e s is it should be evident how
g enetic a l t e r a t i o n s causing changes in normal polypeptide stru ctu re could
70.
influence the chemical, physical and immunological p ro p e r tie s of the pro tein.
Depending on the methods employed certain analytical r e s u l t s will indicate
changes in some p r o p e r tie s which, when viewed collectively, should point to the
fundam ental abnorm ality in basic stru c tu r e .
A L B U M IN G E N E T IC S
E x p re ss io n of the albumin gene is codomin.nt with the two a lle le s both showing
complete p en etran ce. Genetic abnormality of one allele would th en be
controlling the s y n t h e s i s of an abnormal albumin fraction which will c o -e x is t
with th e normal fr a ctio n in the circulation. In humans th i s was the fi r s t
category of t h i s kind of albumin abnormality discovered in the mid 1950's.
About a decade l a t e r th e homozygote condition where both alle le s are abnormal
and code for the same d if fe re n t albumin was described in North American Indian
po pulations. Where bisalbuminaemia was the term used to describe the
h eterozygote s i t u a t io n of two d if fe re n t albumin molecules, alloalbuminaemia is
used to describe both homozygote (one abnormal) and heterozygote (one normal,
one abnormal) albumin conditions. In humans the to t a l albumin concentration in
plasma would be w ithin normal lim its and the two ty p e s of albumin (in the
h e te ro -y g o te situation ) would each comprise about half the to t a l albumin. The
antigenic specificity of the human v arian t fraction s h as been te s te d by
immunoelectrophoretic methods using anti-w hole human serum and anti-hum an
albumin a ntib od ies ra ised in a v ariety of animals (horse, goat, sheep and
rab bit). All th e s e indicate th a t the fractions are immunologically identical to
normal albumin.
V ariant albumin ty p e s have only been described on the s tre n g th of their
71.
e le c tro p h o r e tic behaviour with the b a s i s of the difference thu s being an
a l t e r a t i o n in amino acid make-up such t h a t the overall e lec tric charge changed.
Any amino acid a lte ra tio n t h a t does not involve electric changes will not be
d e te c ta b le e le c tro p h o re tic a lly . The aim ost 100 human v a r i a n ts already described
a re based on a bewilderingly divergent group of electrophone He methods so th a t
some seemingly d if fe re n t v a ri a n ts may in fa c t be the same abnormality
d e m o n s tra te d in d if fe re n t ways. The choice of support medium and pH of
s e p a r a ti o n influences the kind of electrophoretogram t h a t can be obtained and,
fo r human albumin a t l e a s t , a tte m p ts have been made to stan d ard is e th ese two
f a c t o r s fo r th e purpose of detecting and classifying v a ria n t fractions.
Only a few of the human albumin v a rian ts have had th e i r specific abnormality
id e n tif ie d . Of th e s e some had one amino acid s u b s tit u te d in loop 9 and o th e r s
had a s u b s t i t u t i o n in the N -terminal hexapeptide which th e n did not prevent
se c r e tio n b u t prevented the normal p re s e c re tio n p ro te o ly tic splitting off of
t h i s seg m en t. They are th u s abnormal proalbumins in circulation.
S tu d ie s of th e human albumin gene have revealed many more prevalent variatio ns
th a n even tually get e xp ressed in the phenotype albumin molecule, indicating th a t
t h e s e a l t e r a t i o n s must occur to a large e x te n t in the u n tr a n s la te d intron
reg io n s. T his would explain why polymorphism of the albumin gene is estim ated
a t about a 1% occurance but albumin polymorphism is quoted at only about 0,1%.
ISOMERIC FORMS OF THE ALBUMIN MOLECULE
Another source of albumin variation t h a t w arra n ts mention is the existence of
isomeric form s of te p ro tein . Human albumin, when t i t r a t e d with HC1, shows the
appearance of a so called F 'fa s t) form betw een pH 4 - 4,5 which has altered
72.
e le c tro p h o r e tic mobilityi precip ita tion ability, optical c h a ra c te ristic s,
helical co n te n t rev eale d and UV a bsorption. All th e s e changes seem to be
because of a p a r t ia l opening of the albumin molecule exposing groups previously
hidden from e x te r n a l s o lv e n ts . Below pH 4 the molecule becomes fully uncoiled
w ithin th e lim its of i t s bisulphide bonds which c a u se s the threadlike molecules
to in crease the visco sity of their so lu tio n s tremendously, ih is is commonly
called the expanded, E form. The uncoiling seems to be based on the mutual
rep u lsio n of the newly acquired positive charges on the molecule.
On the alkaline side of n eu trality two more isomeric forms e x is t, namely the B
(basic) and A (aged) form. The former is obtained a t pH 9 and is characterized
by a drop in helical con ten t, slower electrophoretic migration and an increase
in th e binding capacity fo r calcium due to the exposure to the outside of the
molecule of numerous hydrogen atoms th a t now become exchangeable. The l a t t e r
aged form is obtained by keeping albumin solutio ns a t low ionic stre n g th a t pH 9
and a t 3°C for 3-4 d ays. This form then has an even slower ele c trophoretic
m igration r a t e and a decreased solubility. These isom ers do not re p r e s e n t the
lim its of the conditions to which albumin can be exposed; t i t r a t i o n can be
conducted rev ersib ly betw een pH 2 and 12 if the time s p e n t at the extrem es is
minimised. Even exposure to 8M urea or 6M guanidinium chloride will not lead to
p erm anent s tru c tu r a l damage. Damage done by a high pH environment seems to be
mostly directed to w a rd s the disulphide bonds w h ereas low pH damage is based on
p eptid e bond cleavage. Heat seems to affect the fa t ty acid free albumin more
than th o se molecules with th ese lipid ligands a ttach ed . Even a f t e r reduction of
all 17 disulphide brid g e s reoxidising them will lead to refolding of the
molecule, s t a r ti n g in each domain simultaneously and independently, with
r e « t c r a t i o n of the m a o r ligand binding capacities to some degree again.
With t h i s as the g en e ra l background within which any d e te c te d albumin v aria tion
can e x e r t i t s e f f e c t and thus make it s e l f evident, one can tu rn to some r e p o r ts
of albumin v a r i a n t s to observe where the specific a lt e r a t io n f i t s into the
la r g e r pictu re.
ANIMAL STUDIES
The a n a ly s is of g en etic material only fairly recently gained momentum with the
advent of many new techniques. The e ffe c ts of specific re s tr i c ti o n enzymes to
d ig e s t the pre pared DNA are d iscu ssed by de Sousa et . 4 (1934) who
described the ex is te n c e of two introm c variant gene fr a c tio n s with the use of
Hae III enzyme which therefore do not become r e p r e s e n te d in the sy n th esis e d
albumin. Lucotte e j J (1984) described the use of Eco R 1 and Hind III
r e s t r i c t i o n endonucle ase enzymes to illu s tra te th ree t y p e s of variant p a t t e r n s
of both the albumin and the alpha feto prote in genes in two inbred s t r a i n s of
r a t s . Gal e t al (1982) also described similar p a t t e r n s obtained in
Sprague-Dawley and Buffalo s t r a i n s of inbred r a t s and followed the inheritance
of c r o s s e s b etw een th ese s t n i n s . Stormout and Suzuki (1963) explained the
ex is te n c e of a p a ir of codominant autosomal a ll e le s for albumin in h o rs e s on the
b a s i s of in heritan ce of phenotypes in two breeds. Juneja e t 4 (1984)
used two dimensional agarose-polyacrylamide ge. e le c tro p h o re sis to ob tain a
p icture of polymorphism in the post-album in region of laboratory ra b b its. They
could link th a t to polymorphism previously re po rte d in th e p re tra n s f e rrin
region, but not t o albumin as such.
O s te r h o ff and co-w orkers have described polymorphism in various wild and
dom estic African animals. They used albumin gene frequencies to indicate the
clear d ifferen ce betw een indigenous and exotic bree is of g o a ts (O sterhoff and
74.
Vitrd-Cox, 1972) and confirmed the functioning of tw o codominant autosomal
a ll e le s in th e albumin t r a n s la t io n in Equidae (O sterho ff, 1969). The d iffe ren t
genetic backgrounds of white and black rh in o cero ss es was also highlighted by
each exhibiting one v a ria n t fractio n of albumin (O sterhoff and Keep, 1970) but
e le c tr o p h o r e s is of serum of the South African b’esbok revealed only onu band
(O sterho ff e t al, 1972). No variatio n could be found in African
e le p h a n t (O sterhoff e t al, 1972) and the same was found for African
b uffalo (O sterhoff e t al, 1970). In the gerbil from Lesotho a high
degree of genetic v ariatio n was found (Maurer et al, 1976) but in the
South A frican impala the operation of two alle le s giving rise to th r e e
ph en oty pes could be d em onstrated (O sterhoff et al, 1972). Studies on
South African c a ttle b reed s also showed t h i s o p eratio n of two d if f e r e n t alleles
(O sterhoff and P re to riu s , 1967).
Worldwide evidence of animal polymorphism is quite abundant. Morera et
al (1983) described two albumin phenotypes in Spanish merino sheep but
Kaminski e t al (1984) found only one type in the ro d e n t A m c a n t u s
nilo ticu s w h ereas Kimura and Yamamoto (1932) re p o r te d the same for the
domestic pigeon. Three phenotypes, caused by two d if fe re n t a ll e le s were
D emonstrated in E a s t African c a ttle (Ashton, 1964) and GusMew icz e t al
(1934) used the genetic polymorphism of t h o r o u g h b r d ho rses to describe blood
line d if fe re n c e s in France and Poland. Maclaren and P e tr a s (1976) iso lated
house mouse albumin and found th a t only one albumin type could be iso late d even
though unwanted polymers had to be removed with the aid of exclusion
chromatography. Comparison of th is albumin to a r a r e variant mouse albumin
showed a difference in electrophoretic mobility only. Haley (1965) designated
the major p ro te in fra ctio n in quail blood albumin and described i t s vai iations
in chicken-quail hybrids.
75 .
In the border a r e a between animal anu man
Lufejr
il'S 4 <
compared the bilirubin
binding capacity of the polymorphic albumin fractions of Tthesus monkeys and that
of American Ind ians. Since t h i s binding did d if fe r in the monkeys he p o stu late d
a s e lectio n in t h a t species t h a t would maintain the polymorphic form. In
In dian s no such binding capacity gain could be d em onstrated.
In humans t r a n s i e n t bisalbuminaemia has been dem onstrated as a p ro teolytic
product in numerous case r e p o r t s (R enverse: e t al, 1984 and Dauth et
al. 1984) of p a n c r e a t it is or pancreatic c y sts and also in th e nephrotic
syndrome (Anmad e t al, 1984). The hete
due to various f a c t o rs was d is c u s s t u
f human
erum albumin
>i969' and Roussoaux e t
al (1982) described a v ariant albumin th a t was unstable upon storage a t 4 C
and a ttain ed the mobility of normal serum albumin. Salaman and Williamson
d isc u ssed the anomalous behaviour of serum albumin when undergoing isoelectric
focusing in the nativ e and Denatured s t a t e s while Spencer and King (1971)
e v alu a te d the isoelectric heterog eneity of bovine plasma albumin. Both th ese
r e p o r t s centre around the so called microheterogeneity of albumin and no
should be taken n ot to confuse changes th us seen with inherited v aria n t t
T illyer e t al (1984) reported on the use of immunofixation methods to
d e te c t genetic albumin v a ria n ts th a t have very similar electrophoretic
m obilities. Au e t i l (1986) more recently isola ted some v arian t human
albumin and in v e s tig a te d i t s p ro p e rtie s. They found sim ilar antigenic
re actio n s , sim ilar binding p ro p e r tie s and similar uptake capacities for dyes
when comparing variant to n ativ e albumin.
THE PRESENT STUDY
Regarding the degree to which the r e c u l ts
f the lion albumin in v e s tig a tio n s
a g ree with the wealth of information d ealt with above care has to be exercised.
From th e e le c tro p h o r e s is r e s u l t s of the various techniques one can s t a t e th a t
th e mobility difference betw een the two fraction s i s maintained in sp ite of
changes in sample collection, sample handling and donor d ifferences. The SDS
e le c tro p h o r e s is techniques would se p a r a te the p ro te in s on the b a sis of size much
more than charge and th o se r e s u l t s indicate the d ifferen ce betw een the albumin
f r a c ti o n s to be one of charge. The inability of the exclusion columns to
s e p a r a t e the two f r a c tio n s would also point to a very similar molecular mass
stre n g th e n in g the concept th a t the charge is the most obvious distinguishing
f e a t u r e . Thus charge could be created by a specific ligand binding or by some
ge n e tic coding for two d if fe re n t ty p e s of sequences. Regarding the f i r s t
p o s s ib ility one can s t a t e th a t such binding has to be extremely tig h t so th a t no
a l t e r a t i o n in sep aratin g condition or in donor s e lectio n would a ff e c t it in any
way. Furtherm ore, the probability th a t ligand binding can cause such a dramatic
d iffe ren ce h a s never been recorded. Microheterogeneity could, however, r e s u lt
from such ligand a tta c h m e n ts and th es e were indeed dem onstrated by the
is o e le c tric focusing r e s u l t s , yielding an albumin zone ra t h e r than single bands.
The second po ssib ility seem s th erefo re a more fea sab le explanation. Only amino
acid sequencing can r e v e a l the exact natu re of the a lt e r a t io n but similar human
a n a ly s is h as shown the su b s titu tio n to involve only one amino acid, thereby
changing the overall charge slightly but not affecting molecular mass. This
a l t e r a t i o n h a s to be genetically determined but car, a ff e c t the polypeptide
p ro per or the leader hexapeptide of proalbumin. The fact th a t th is su b s titu tio n
only involves one amino acid also explains why the immunological character is
no t affec ted ; the chances of altering the specific antigenic zone are very
r mote and it h a s n ot been re p o rte d for any spec ies. Any analytical procedure
b a s j on anything e ls e but molecular charge will fail to re v e a l the variant
fr a c tio n .
The genetic background to th i s is difficult to explain since the normal two
codominant gene s i tu a t io n would re s u lt in a 50% occurence of each fraction. In
t h i s case the r a t io is very d if fe re n t, something also repo rted in one human
v a r i a n t fraction a s well as in the repo rts on rh in o cero ss es by O sterh off. I t is
p o ss ib le th a t the genetically determined a lt e r a t io n is in the proalbumin region
s o t h a t the sp littin g o ff continues in the circulation, keeping the variant
fr a c tio n at a lower concentration than the normal fraction. Another
p o s s ib ility , already mentioned by O sterhoff in the o ther animal re s u lts , is th a t
th e tw o -a lle le system is most probably too sim plistic and can be extended to
include other gene p a ir s . This was already offe red a s an explanation for the
ex is te n c e of five phenotypes of plasma p ro te in and one abnormal allele here
could th en code fo r a v a ria n t fraction which will e x is t a t a lower concentration
th a n 50% of the to t a l albumin.
In humans the alloalbuminaemia occurs only a t a very low frequency, making an
argument for naming the condition a spo ntaneous mutation or calling it a
polymorphic v ariatio n . The l a t t e r would involve some se lection providing a
b e n e f i t to the animal. In lions the frequency is 100% so t h a t th is is
d efin itely b a-ed «n n atural selection. The p ossible b en e fit could lie in the
d if f e r e n t binding p ro p e r tie s of the variant forms, something hinted at in
p revious animal s tu d ie s .
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Author Joo ste C P
Name of thesis Isolation and Limited Characterisation of Lion - Panthera Leo Albumin 1987
PUBLISHER:
University of the Witwatersrand, Johannesburg
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