Lanreotide, Somatost

Low Concentration Structural Dynamics of Lanreotide and
Somatostatin-14
Belen Hernandez,1 Yves-Marie Co€ıc,2 Bruno Baron,3 Sergei G. Kruglik,4,5 Fernando Pfl€
uger,1
6
7
1
Regis Cohen, Claude Carelli, Mahmoud Ghomi
1
Groupe de Biophysique Mole culaire, UFR Sante -Me decine-Biologie Humaine, Universite Paris 13, Sorbonne Paris Cite , 74
rue Marcel Cachin, 93017 Bobigny cedex, France
2
Institut Pasteur, Unite de Chimie des Biomole cules, UMR 3523, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France
3
Institut Pasteur, Plate-Forme de Biophysique de Macromole cules et de leurs Interactions, 25, Rue du Docteur Roux, 75724
Paris Cedex 15, France
4
Sorbonne Universite s, UPMC Universite Paris 06, UMR 8237, Laboratoire Jean Perrin, F-75005 Paris, France
5
CNRS, UMR 8237, Laboratoire Jean-Perrin, F-75005 Paris, France
6
Service d’Endocrinologie, Centre Hospitalier de Saint-Denis, 2 Rue du Docteur Delafontaine, 93200 Saint-Denis, France
7
Regulaxis, Parc Scientifique Biocitech, 102 avenue Gaston Roussel, 93230 Romainville, France
Received 18 February 2014; revised 21 March 2014; accepted 26 March 2014
Published online 11 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22491
ABSTRACT:
Lanreotide, a synthetic cyclic octapeptide, analogue of
the peptide hormone somatostatin-14 (SST-14), is
routinely used as a long-acting medication in the
management of neuroendocrine tumors. Despite its
therapeutic importance, low concentration structural
data is still lacking for lanreotide. In fact, the major
part of the previous structural investigations were
focused on the remarkable aggregation properties of
this peptide, appearing at high concentrations (>5
mM). Here, we have applied three optical spectroscopic techniques, i.e. fluorescence, circular dichroism
and Raman scattering, for analyzing the structural
dynamics at the concentrations below 5 mM, where
lanreotide exists either in a monomer state or at the
first stages of aggregation. The obtained data from
lanreotide were discussed through their comparison
following conclusions: (i) The central D-Trp residue,
forming with its adjacent Lys the main receptor
interacting part of lanreotide, keeps a constant high
rotational freedom whatever the environment (water,
water/methanol, methanol). (ii) A solvent-dependent
tight b-turn, belonging to the type-II’ family, is
revealed in lanreotide. (iii) Raman data analyzed by
band decomposition in the amide (I and III) regions
allowed estimation of different secondary structural
elements within the millimolar range. Interestingly,
the applied protocol shows a perfect agreement
between the structural features provided by the amide
C 2014 Wiley PeriI and amide III Raman markers. V
odicals, Inc. Biopolymers 101: 1019–1028, 2014.
Keywords: lanreotide; type-II’ b-turn; somatostatin-14;
structural dynamics; fluorescence; circular dichroism;
Raman scattering
with those collected from SST-14, leading us to the
Correspondence to: Mahmoud Ghomi; e-mail: mahmoud.ghomi@univ-paris13.fr
C 2014 Wiley Periodicals, Inc.
V
Biopolymers Volume 101 / Number 10
This article was originally published online as an accepted
preprint. The “Published Online” date corresponds to the
preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
1019
1020
Hernandez et al.
INTRODUCTION
S
omatostatin-14 (SST-14) (Figure 1) is a natural
tetradecapeptide hormone which takes part in the
inhibition of the release of growth hormone,
thyroid-stimulating hormone, insulin and glucagon.1–6 Considering the prominent therapeutic role
of this peptide hormone and its short plasma half life (<3
min), an important effort has been directed toward the elaboration of efficient synthetic anlaogues.7,8 Among them, lanreotide (or BIM 23014) (Figure 1) is currently used as
therapy in human diseases.9 From the structural point of
view, the cyclic structure of lanreotide is maintained by a
unique Cys2-Cys7 disulfide linkage, similar to the Cys3-Cys14
one in SST-14. Moreover, two D-stereoisomers are incorporated in lanreotide, D-Nal1 and D-Trp4, where Nal stands for
naphtylalanine. The Trp!D-Trp substitution was previously
shown to have an important effect on the increase of the biological activity of SST-14 octapeptide analogues.3,5
During the last decade, the spectacular aggregation features
of lanreotide have given rise to a series of valuable investigations
by means of imaging and spectroscopic techniques.10–16 In this
framework, the propensity of this short cyclic peptide to provide multiscale nano-objects upon increasing concentration has
been evidenced. In fact, three successive stages of aggregation
were revealed,15 (i) monomer!dimer association (Kd 5
mM), followed by the stabilization of (ii) open ribbons formed
by higher order association (Kd 15 mM) which are finally
closed in (iii) nanotubes (Kd 21 mM). At a final step, the
formed nanotubes can be packed into a liquid crystalline hexagonal phase. However, apart a few fluorescence measurements,13 no consistent set of data is available on the structural
properties of lanreotide at low concentrations, i.e. 5 mM. In
parallel, the analysis of SST-14 aggregation features have evidenced the formation of nanofibrils with controlled diameters
at the concentrations above 20 mM,17 followed by that of
nanofibers above 65 mM.18 The ionic strength effect on the
formation of nanofibrils and nanofibers, as well as on the compaction of nanofibers, giving rise to spherulite-like amyloid
droplets, has also been described.17,18 At lower concentrations
(below 20 mM), the structural dynamics of SST-14 in aqueous
solution is consistent with an unordered loop which gradually
looses its flexibility upon increasing concentration from submillimolar to millimolar concentrations.19
In the present article, we report our attempts to get insight
into the low concentration structural dynamics of lanreotide,
compared to that of SST-14, by means of three complementary
techniques, fluorescence, circular dichroism (CD) and Raman
scattering. A special attention has been paid to the effect of
environment (ionic strength and solvent permittivity) on the
structural features of both peptides.
FIGURE 1 Amino acid composition of the tetraoctapeptide hormone SST-14 (right) and its octapeptide analogue lanreotide (left).
Amino acids are numbered from Nter to Cter.
RESULTS AND DISCUSSION
High Rotational Freedom of the Trp Residue
The first fluorescence data of lanreotide obtained at 10 lM,13
showing a dominant contribution of the D-Trp4 residue, have
provided an anisotropy value of <r> 5 0.02, consistent with
the high rotational freedom of the latter residue in aqueous
media. Further fluorescence measurements on more concentrated samples could manifest a drastic anisotropy increase
(10 fold) due to the aggregation in the 15–21 mM concentration interval.15 Figure 2 shows the emission spectrum of lanreotide and SST-14 at 25 lM in three different environments.
In both peptides, the Trp signal, characterized by a large band
at 350 nm in aqueous solution, is downshifted to 340 nm
in MeOH. A similar effect has been reported in SST-14 in
going from aqueous to lipid (with a lower electric constant
compared to water) environment.20,21 Normalized fluorescence
is decreased in water compared to methanol, with a rate of
10% in lanreotide (Figure 2a) and 50% in SST-14 (Figure
2b). In contrast, the ionic strength is shown to have a minor
effect on the fluorescence signal. The measured anisotropy values at 25 lM are reported in Table I for both peptides. Previously, a low L-Trp8 anisotropy (<r>0.03) was reported (at
unknown concentration) in SST-14.21 This value, being three
times larger than that estimated presently at 25 lM
(<r>0.011) (Table I), might correspond to a higher concentration. In MeOH, an approximately twofold increase of the
<r> value is estimated for SST-14 (Table I). A similar behavior
has been reported for SST-14 in the presence of lipids.21 No
similar effect has been revealed for the D-Trp4 residue involved
in lanreotide, for which a rather constant anisotropy value is
estimated in all environments, close to the highest value measured in methanol for SST-14 (Table I).
Biopolymers
Structural Dynamics of Lanreotide and Somatostatin-14
1021
FIGURE 2 Fluorescence spectra recorded at 25 lM in H2O, H2O/150 mM NaCl and methanol.
The concentration corresponds to 0.04 and 0.027 g L21 for SST-14 and lanreotide, respectively.
Because of the presence of a Tyr residue in its chemical composition, lanreotide was excited at 295
nm instead of at 290 nm in SST-14.
A Type-II’ b-turn is Revealed in Lanreotide
Up to now, no CD data was reported forr lanreotide. Figures
3a and 3a0 present the CD spectra of this peptide at 100 lM.
No perceptible change is observed in H2O solution CD spectra
in the 25–200 lM range (data not shown). In all environments,
these spectra are characterized by a quite resolved negative
doublet located within the 200–230 nm range, followed by a
weak positive band at 239 nm. Recently, CD analysis of
octreotide (another SST-14 octapeptide analogue) has allowed
assignment of this particular CD fingerprint to a type-II’ bturn.22 It should be emphasized that the latter reverse folding
had been first evidenced in octreotide by means of crystal23
and solution (DMSO) NMR24 data. Figures 3a and 3a0 show
that the ellipticity ratio of the above mentioned negative doublet (namely U2/U1, where U2 and U1 refer to the ellipcities of
the higher and lower wavelength components of the doublet,
respectively), along with the position of the higher wavelength
component, are environment-dependent. More precisely,
U2/U1, being close to unity in water, is progressively lowered to
0.8 in water/methanol mixture (Figure 3a), and finally to 0.6 in
pure methanol (Figure 3a0 ). The previously reported CD studies performed on ten gramicidin S based cyclic peptides,
adopting type-II’ b-turns,25 have shown that the U2/U1 ratio
can be taken as an indicator of the turn stability. In other
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words, a U2/U1 ratio close to unity is consistent with an
unusually stable b-turn, whereas a value close to 0.5 reveals a
moderately stable folding. Based on this criterion, it can be
concluded that lanreotide, adopting a very stable turn in water,
looses gradually its tightness when it is found in lower dielectric constant media (water/methanol or methanol). As far as
SST-14 is concerned, the presence of a single negative band at
203 nm (Figures 3b and 3b0 ) in its CD spectrum, compatible
with a U2/U1 0, proves the very flexible loop formed in this
cyclic peptide.19 As previously reported, no significant change
has been observed in SST-14 CD spectra within a large submillimolar concentration interval (100–500 lM).19 However,
Table I Fluorescence Anisotropy Values, <r>, Measured in
Different Environmentsa,b
Lanreotide
SST-14
a
Pure Water
Water/150 mM NaCl
Methanol
0.022
0.011
0.022
0.011
0.026
0.023
All measurements were performed at 25 lM. This concentration corresponds to 0.027 and 0.04 mg mL21 for lanreotide and SST-14, respectively.
Each value corresponds to the average of 60 measurements.
b
Identical anisotropy values were obtained for three excitation wavelengths at 275, 290. and 295 nm.
1022
Hernandez et al.
FIGURE 3 CD spectra recorded at 100 lM in water (blue), water/methanol (50%/50% mixture,
pink) and methanol (red). The selected concentration corresponds to 0.16 and 0.11 mg mL21 for
SST-14 and lanreotide, respectively.
other CD data (recorded at unknown concentration) have
revealed the presence of a weak shoulder at 215 nm, consistent with a structuring trend of SST-14 in aqueous media.
Nevertheless, the negative doublet fingerprint of this peptide
hormone, observed in a lipid environment,21 proves undoubtedly its structuring tendency in hydrophobic media.
Raman Markers from Tight and Loose Turns
Previous reports on lanreotide aggregation dynamics have provided limited Raman data. They were basically discussed in the
specific spectral regions where the environment of the aromatic
residues (Tyr and Trp), as well as the disulfide linkage conformation, could be explored.10,11,13,14 An elevated range of con-
centrations, i.e. 10–150 mM, was used in the above
mentioned experiments. We have recently reported the Raman
data of SST-14 in the 5–20 mM concentration range,19 showing a clear structuring effect of the corresponding large size
loop upon increasing concentration. For our discussion on the
Raman spectra of both peptides, we have selected a common
concentration, i.e. 5 mM. Precisely, this concentration corresponds to the previously reported Kd value of the first aggregation step (monomer–dimer) in lanreotide.15 To remove any
doubt about a possible interference of monomer and dimer
spectra, we have checked (data not shown) that no notable
change was observed in the Raman data obtained in the 1–10
mM range. This allows us to conclude that the aggregation
process does not induce any perceptible conformational
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Structural Dynamics of Lanreotide and Somatostatin-14
1023
FIGURE 4 Subtraction of the solvent contribution from the Raman spectra observed in lanreotide dissolved in H2O (a) and D2O (b), Red trace spectra are from the solutions containing lanreotide. Blue trace spectra are those obtained from the solvents. Green traces correspond to the solvent
subtracted spectra of lanreotide. These spectra correspond to 5 mM (5.5 mg/mL) peptide
concentration.
change in lanreotide. In other words, the dimer formation
must be due to the aromatic/hydrophobic interactions between
the turn residues without altering the turn conformation. In
SST-14, the selected concentration is located well below the
concentration threshold (20 mM) for initiating the fibrillation process in aqueous solution.17,18
The extraction of structural information from the amide I
(1700–1600 cm21) and amide III (1320–1220 cm21) regions
requires, however, a careful post-record treatment of the
observed data, avoiding any possible artifact arising from the
Biopolymers
buffer subtraction. In addition, the assignment of the observed
bands to the amide vibrations needs the collection of Raman
data in heavy water. For instance, D2O Raman spectra confirm
the assignment of the amide III markers by their complete vanishing due to the NH!ND substitution in a peptide backbone.19,26,27 To check all these crucial points, we present in
Figures 4a and 4b the Raman spectra obtained from (i) the
solution containing lanreotide (red trace), (ii) the solvent contribution (blue trace), and finally (iii) the spectrum obtained
from the subtraction of the latter two spectra (green trace).
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Hernandez et al.
FIGURE 5 Solvent subtracted Raman spectra of lanreotide observed in the 1750-350 cm21 spectral region. (a) and (b) refer to the Raman spectra obtained from H2O and D2O solutions, respectively. Tentative assignments of the observed Raman bands are also reported. These spectra
correspond to 5 mM (5.5 mg/mL) peptide concentration.
The final spectra are displayed in Figures 5a and 5b, along with
the tentative assignment of their main bands. Because of its
aromatic character, D-Nal1 residue gives rise to eight strong/
middle bands observed in the selected spectral range (wavenumbers reported in pink), among which only the two bands
located at 1579 and 518 cm21 were commented in the previous reports on lanreotide.10,13,14 The analysis of the amide
(I and III) regions by band decomposition is obviously based
on the choice of significant components. This goal has been
achieved by means of our previously reported Raman spectra
Table II Determination of the Populations of Different Secondary Structure Elements by Band Decomposition
Lanreotide
Amide I (H2O)
Markers
1692 (Random)
1678 (Turn)
1661 (b-strand)
1652 (Turn)
Total Turn
Total b-strand
Total Random
Sum of areas
Lanreotide
Amide I’ (D2O)
SST-14a
Amide I (H2O)
Lanreotide
Amide III (H2O)
Area (Width)
Markers
Area (Width)
Markers
Area (Width)
10 (14)
48 (16)
30 (16)
12 (13)
1680 (Random)
1669 (Turn)
1656 (b-strand)
1646 (Turn)
10 (15)
47 (17)
30 (17)
13 (12)
1303 (Turn)
1289 (Turn)
1265 (Random)
1252 (Turn)
1233 (b-strand)
11 (13)
5 (12)
12 (13)
43 (17)
29 (18)
59
29
12
100
60
30
10
100
60
30
10
100
Area (Width)
1689 (Random)
1678 (Turn)
1665 (b-strand)
1652 (Turn)
48 (19)
30 (17)
12 (12)
10 (14)
40
12
48
100
All reported values refer to the spectra recorded at 5 mM, corresponding to 5.5 and 8.2 mg mL21 for lanreotide and SST-14, respectively.
a
Taken from our previously published report on SST-14.19
Marker band wavenumbers, as well as their widths, are in cm21; their normalized areas are in % (accuracy 65%, see text for details).
Biopolymers
Structural Dynamics of Lanreotide and Somatostatin-14
1025
FIGURE 6 Band decomposition in the amide regions of the Raman spectra obtained from lanreotide aqueous samples at 5 mM. (a) Amide I region observed in H2O solution. (b) Amide I’ region
observed in D2O solution. (c) Amide III region observed in H2O solution. Circles correspond to
the sum of the components used in band decomposition.
on other short peptides, forming b-strands27 and turns19,22 in
aqueous solution (see below for more details). More precisely,
the characteristic components assigned to b-strands, b-turns
and disordered chains (with a half-width not exceeding 20
cm21), were introduced as initial guess in the first stage of
band decomposition. Their wavenumbers and widths were
slightly varied in order to improve the fit to the observed spectrum. A minimum number of components (without complete
overlap) were used. Several trials (at least three) of band
decomposition in each region (amide I and amide III) allowed
us to evaluate the accuracy on the estimation of populations
relative to different secondary structural elements, see below
and Table II for details.
Amide I Region. Despite the presence of the strong band at
1634 cm21 arising from D-Nal1 residue, we could analyze the
amide I (Figure 6a) and amide I’ (observed in D2O, Figure 6b)
regions. Upon backbone N-deuteration, the amide I components manifest a 5- to- 12 cm21 downshift, in agreement with
previous observations in peptides19,22,26,27 and proteins.28 Considering our previously reported data on SST-1419 and octreotide,22 four components assigned to turn, b-strand and
random chain, were used for band decomposition (Table II).
Both regions (amide I and amide I’) provide similar populations of secondary structural elements (Table II). This result
seems interesting because it reveals that the decoupling of the
C@O bond-stretch from the NAH bending motions, due to
the NAH!NAD substitution in the peptide backbone, does
not perturb the structural analysis in the amide I/amide I’
Biopolymers
region. Even the bandwidths of the components used in these
two regions are fairly comparable. However, as already
reported in other short cyclic peptides,29 possible intra-chain
coupling of the C@O bond-stretch motions, affecting the estimated populations of the turn and b-strand elements, should
also be taken into consideration.
Amide III Region. Our recent work on octreotide22 allowed us
to propose a protocol for an accurate analysis of the amide III
region corresponding to a type-II’ b-turn. In fact, the last decade
has seen the report of a series of valuable investigations of the
amide (I, II, and III) regions of short and large size peptides by
means of ultraviolet resonance Raman (UVRR) spectroscopy.30–33 Based on these works, useful empirical equations,
relating the amide III wavenumber of a given residue to its backbone w angle, were established.34 It should be recalled that a tight
b-turn is a four residues folding, in which the two middle residues, referred to as i 1 1 and i 1 2, adopt special / and w angles,
while i and i 1 3 residues keep the torsion angles close to those
of a standard b-strand. On the basis of the above mentioned
empirical relations, we could assign the components at 1303
and 1252 cm21 to the i 1 1 and i 1 2 residues of a type-II’
b-turn.22 On the other hand, classical Raman spectra have previously assigned the two components at 1265 and 1233 cm21
to random chain and b-strands, respectively.19,22,26,27 Finally, the
fifth component of this region appearing at 1289 cm21 has
been recognized as a turn marker, since the earliest Raman studies.35 Figure 6c shows the amide III band decomposition in
lanreotide. The agreement between the populations of turn,
1026
Hernandez et al.
b-strand and random chain elements estimated by the amide III
and amide I markers, is to be emphasized (Table II).
It is worth of noting that the above mentioned Raman
markers can clearly evidence the flexibility/rigidity of both
cyclic peptides. The populations indicated in Table II are consistent with a structured loop in lanreotide, as its random
chain contribution represents only 10%. In contrast, in SST14, a random population of 50% confirms the loose type
folding of this peptide.19
Disulfide Bond Stretch. The disulfide linkage conformational flexibility is generally analyzed by the Raman markers
observed in the 550–500 cm21 spectral region, mainly due
to the SAS bond stretch motions, namely m(SAS). The
Raman spectral shape of lanreotide at 5 mM (Figure 5a) is
similar to that previously reported at a 20 fold higher concentration.14 It has been shown that only the Raman band at
505 cm21 can be assigned to m(SAS) vibration.14 In fact,
upon D-Nal!D-Phe substitution, the narrow Raman band
at 518 cm21 (Figure 5) could be entirely assigned to the DNal1 residue, discarding any possible overlap of this band
with another type of m(SAS) marker generally observed at
520 cm21. As previously shown,19 SST-14 reveals at 5 mM,
all the three known m(SAS) markers at 505, 520, and
540 cm21. This proves the high conformational flexibility
of the disulfide bridge in SST-14. However, upon increasing
concentration to 20 mM, the 505 cm21 marker becomes
the major component in SST-14 Raman spectrum, presumably due to the loss of flexibility of this peptide due to its
increasing aggregation. All these evidences, collected from
lanreotide (with a tight turn) and SST-14 (with a loose
turn), confirm the correlation existing between the turn and
disulfide conformational flexibilities.
General Remarks on the Structural Features of
Lanreotide and SST-14-Possible Relations with Their
Biological Activities
The type-II’ b-turn folding of lanreotide is certainly nucleated
by the heterochiral -D-Trp4-Lys5- pair, i.e. i 1 1 and i 1 2 residues of the turn (Figure 1).25 Its concentration-independent
stability is presumably reinforced by the hydrophobic interactions of the residues involved in the loop, particularly those
between the aliphatic side chains of the adjacent -Lys5-Val6pair (i 1 2 and i 1 3 turn residues), and a strong stabilizing Hbonding between the Tyr3 and Val5 (i and i 1 3) backbones.
CD spectra of lanreotide could prove the high stability of its
type-II’b-turn at the concentrations as low as 25 lM. A completely different situation has been revealed for SST-14 because
of its large size loop. In fact, the presence of two charged (Lys4
and Lys9) and four polar (Asn5, Thr10, Thr12, Ser13) residues in
the cyclic part (Figure 1) favor the interactions with the surrounding water molecules, and consequently the formation of
a loose scaffold. The SST-14 flexibility, evidenced by our present and previously reported spectroscopic data,19 contradicts
the conclusions derived from the earliest CD data, suggesting a
rigid b-sheet pleated structure for this peptide hormone.36,37
In contrast, our results corroborate the previous NMR data,
consistent with the existence of a loose turn in SST-14.38
Nevertheless, in both peptides, the Trp residue (L-Trp8 in SST14 or D-Trp4 in lanreotide) keeps a high mobility, necessary
for their interactions with SST-14 receptors.38 In this framework, the high affinity of lanreotide for the two (out of five)
SST-14 receptors, referred to as SSTR2 and SSTR5, is be
stressed.39 This feature can be better understood by considering
the very stable reverse type folding of this peptide. In contrast,
SST-14 with its loose turn, can moderately interact with all the
five receptors (SSTR1, . . ., SSTR5), presumably through an
induced fit process.40 Raman data have also shown the close
relationship between the turn and disulfide linkage conformational flexibilities. Previous observations on other SST-14 octapeptide analogues have shown that a change in the disulfide
linkage length has a direct consequence on the turn conformation, as well as on the binding affinity to SSTRs.41 Inversely, an
increase of the turn length, induced by conjugating an additional methylene group to its backbone, was shown to change
the conformational flexibility, as well as the receptor binding
affinity.42
CONCLUSION
Differences between the low concentration structural features
of lanreotide and SST-14 were evidenced through the combined use of three optical spectroscopic techniques, i.e. fluorescence, circular dichroism and Raman scattering. We would like
to stress the capability of the classical Raman spectra for providing structural information on the short size peptides
through the amide (I and III) regions.19,22,26,27,43,44 Particularly,
the ability of the amide III Raman markers to confirm the reliability of the structural information deduced from the amide I
(or amide I’) region, as confirmed here for lanreotide, should
be emphasized.
MATERIALS AND METHODS
Synthesis, Purity, and Sample Preparation
Lyophilized samples of lanreotide/acetate salt were provided from
IPSEN. Purity control of the peptide was assessed by RP-HPLC on an
Agilent (Santa Clara, CA) 1100 Series liquid chromatograph and
Biopolymers
Structural Dynamics of Lanreotide and Somatostatin-14
monitored with a photodiode array detector by absorbance at 230
nm. A linear gradient (0,5%/min) of acetonitrile in 0.08% aqueous
trifluoroacetic acid was applied over 20 min at a 0.35 mL min21 flow
rate on an Aeris PEPTIDE 3.6 lm XB-C18 100 3 2.10 mm (Phenomenex, Le Pecq, France). UV chromatograms confirm the high purity
(>99%) of the peptides. Mass spectrometry was carried out on a
quadrupole time of flight (Q-TOF) Micro mass spectrometer (Waters,
Manchester, UK) equipped with a Z-spray API source and calibrated
with a phosphoric acid calibration solution. Capillary sample cone
and extraction cone voltages were set at 3 kV, 30 V and 10 V, respectively. Source and desolvation temperatures were set at 80 and 250 C,
respectively. Data were acquired by scanning over the m/z range 150–
2000 at a scan rate of 1 s and an interscan delay of 0.1 s. Lanreotide
was dissolved in a mixture of water/methanol/acetic acid 49.5:49.5:1
v/v/v at a concentration of 100 ng lL21 and analyzed in positive-ion
mode by infusion at a flow rate of 5 lL min21. Sixty spectra were
combined and the resultant raw multicharged spectra were processed
using the MaxEnt 3 deconvolution algorithm embedded in the Masslynx software. The experimental data were consistent with the
expected mass of lanreotide, i.e. monoisotopic [M1H]1 1096.4743 d,
observed 1096,4716 d.
Sodium chloride (Merck, purity > 99.5%), Methanol (Carlo Erba,
ACS-ISO for analysis), Fresh pure water (Millipore filtration system),
D2O (Euriso-top, Saclay, France, 100% purity) were used for preparing
the used samples. Stock solutions were prepared by dissolving the
lyophilized peptide in pure water (or in pure D2O). For CD and
fluorescence experiments, the stock solution at 5 mM was diluted in
different solvents (H2O, H2O/150 mM NaCl, H2O/MeOH and MeOH)
to reach the final concentrations within the 25–500 lM range. For
Raman experiments in H2O, the dilution was undertaken from a stock
solution at 20 mM to obtain lower concentrations at 10, 5, 2.5, and
1 mM.
Spectroscopic Protocols
Fluorescence Spectra were recorded on a PTI Quanta-Master
QM4CW spectrofluorometer (Lawrenceville, NJ) at 25 C using a 10
mm pathlength quartz cell. Excitation and emission bandwidths were
set to 1 and 5 nm, respectively. Corresponding values for anisotropy
measurements were 5 and 15 nm, respectively. Fluorescence anisot2GIVH
ropy, <r>, was estimated according to the relation: hri5 IIVVVV12GI
,
VH
IHV
where the G factor is given by: G5 IHH , and I denotes the polarized
intensity corresponding to the vertical (V) and horizontal (H) polarization states (with respect to the plane including the incident and fluorescence beams) of the excitation (first subscript) and emission
(second subscript) radiations.45
CD Spectra were analyzed on a JASCO J-810 spectrophotometer
within the 190–300 nm spectral region (path length 1 mm, spectral
resolution 0.2 nm). Each spectrum was recorded with a speed of 100
nm min21, and corresponds to an average of five scans. To facilitate
the comparison of CD spectra, their normalized ellipticity was
expressed in deg cm22 dmol21.
Stokes Raman spectra were recorded at room temperature. Excitation was made by means of the 488 nm line of an Ar1 laser (Spectra
Physics), 200 mW power at the sample. Scattered light at right angle
was analyzed on a Jobin-Yvon T64000 (single spectrograph configuration, 1200 grooves/mm holographic grating and a holographic notch
filter). Raman data (1200 s acquisition time for each spectrum) were
collected on a liquid nitrogen cooled CCD detection system (Spec-
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1027
trum One, Jobin-Yvon), effective spectral slit width was set to about 5
cm21. GRAMS/32 software (Galactic Industries) was used for buffer
subtraction and smoothing of observed spectra. The analysis of the
amide I and amide III regions was performed by curve fitting using
pseudo Voigt (Gaussian1Lorentzian) functions, with the Lorentzian
contribution kept equal to, or greater than 50%. Final presentation of
CD and Raman spectra was achieved by means of SigmaPlot package
(Systat Software, Point Richmond, CA).
Likewise, sample preparation, purity control and spectroscopic
protocols concerning SST-14, were described in our previous report
devoted to this peptide hormone.19
The authors thank IPSEN (Courtabœuf, France) to have generously provided the lanreotide samples used in this work.
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