`Insulinotropin in the Solid State: a-Helix to @-Sheet Conversion Induced by
`Phenol and/or by High Shear Force
`
`YESOOK KIM'^, CAROL A. ROSE', YONGLIANG L I ~ , YUKIHIRO OZAKI~~, GEETA DATTA*, AND ANTHONY T. Tugx
`Received February 18, 1994, from the *Pharmaceutical Research and Development, Central Research Division, Pfizer, Inc.,
`Groton, CT 06340, #Department of Chemistry, Kwansei Gakuin University, Nishinomiya 662, Japan, and *Department of
`Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523.
`Accepted for publication May 9, 1994@
`
`I
`
`15
`
`10
`
`25
`
`30
`
`5
`
`20
`
`His - Ala - Glu - Gly - Thr - Phe - Thr -
`Ser - Asp - Val - Ser - Ser - Tyr - Leu -
`Glu - Gly - Gln - Ala - Ala - Lys - Glu -
`Phe - Ile - Ala - Trp - Leu - Val - Lys -
`Gly - Arg - Gly
`
`Abstract 0 Insulinotropin (glucagon-like peptide I) is a peptide
`containing 31 amino acid residues. I t stimulates the secretion of the
`hormone insulin. The solubility of this peptide is highly dependent on
`its environment and the treatment that it has undergone. For instance,
`synthetic insulinotropin is highly soluble in neutral phosphate-buffered
`saline (1 mg/ml). However, the application of shear force by stirring
`renders it extremely insoluble (1 hg/mL). This property may be
`explained in terms of a change in peptide secondary structure with
`no alteration in primary structure.
`I n order to understand this
`phenomenon, FT-IR and near-IR FT-Raman were employed to examine
`four samples prepared under different experimental conditions. I t
`was found that solubility decreases as the a-helix is converted to an
`antiparallel &sheet structure.
`
`Introduction
`
`Proteins and peptides are often most soluble under conditions
`mimicking their natural environment. When they are exposed
`to unnatural environments, they tend to form precipitates or
`aggregates as a result of decreasing solubility. The physical basis
`for protein insolubility is still unclear. However, it has been
`proposed that the hydrophobicity of amino acids,14 the primary
`structure, and the peptide backbone conformation7-* all con-
`tribute toward the solubility of the protein. The peptide
`backbone conformation may be indicative of the solubility of
`the protein. Many studies have suggested that protein insolu-
`bility may depend on the content of the p-sheet structure. For
`instance, insulin forms insoluble fibrils when heated in acid.
`Structural studies carried out by Burke and Rougvieg showed
`that the fibrils had a cross-p-structure. It is well-known that a
`@-peptide is a major component of amyloid deposits in Alz-
`heimer's disease.1&l2 Analysis of a protein that was precipitated
`by salting-out also demonstrated a correlation between increased
`&sheet structure and decreased s01ubility.l~
`Glucagon-like peptide I (insulinotropin) is a 31-amino acid
`peptide,lP17 the primary sequence of which is shown in Figure
`1. The solubility of insulinotropin amorphous materials varies
`from 1 mg/mL to 1 hg/mL in neutral pH phosphate-buffered
`saline depending on the method of preparation. For a better
`understanding of what causes the dramatic changes in solubility
`of insulinotropin, characterization of the secondary and tertiary
`structures of these solid materials is essential. FT-IR and Raman
`spectroscopy are the most widely used techniques to characterize
`protein structures in the solid state, especially when the material
`is amorphous, In order to examine the relationship of the p-sheet
`content to solubility, the structure of insulinotropin was studied
`in its highly soluble form and in its insoluble form using FT-IR
`and Raman spectroscopy.
`
`e Abstract published in Advance ACS Abstracts, June 15,1994.
`
`Figure 1-Primary structure of insulinotropin.
`
`Our study showed that the percentage of &sheet conformers
`played a role in determining its solubility in aqueous media.
`
`Experimental Section
`Materials and Methods-Insulinotropin
`(lot #'501713) used through-
`out this study was synthesized by solid-state peptide synthesis and was
`obtained from Pfizer, Inc. Dulbecco's phosphate-buffered saline (PBS)
`was purchased from Gibco (Life Technologies, Inc.). Phenol (fused,
`USP grade) was obtained from J. T. Baker.
`Sample A was the original synthetic insulinotropin, which was readily
`soluble. A 1 mg/mL insulinotropin solution in water for injection was
`stirred vigorously with a magnetic stir bar for 24 h to form aggregates.
`The aggregates were isolated by centrifuging the sample to pellet the
`solids and removing the supernatant. These isolated solids were labeled
`as sample B. For the preparation of sample C, a 2 mg/mL insulinotropin
`solution in PBS and a 4.4 mg/mL phenol solution in PBS were combined
`in a 1:l volume ratio in a glass vial. The vial contents were inverted
`several times to mix the two solutions thoroughly. A precipitate formed
`immediately. However, the vial was sealed and kept at ambient
`temperature for 24 h to ensure complete precipitate formation. The
`precipitates were isolated by centrifuging the sample to pellet the solids
`and removing the supernatant. These isolated solids were labeled as
`sample C. Sample D was prepared in the same manner as sample C
`except that its contents were stirred vigorously for 24 h with a magnetic
`stir bar prior to centrifugation.
`The equilibrium solubility for samples A-D was determined at room
`temperature by dispersing an excess amount of solid into PBS with a
`vortex mixer for approximately 10 s. This dispersion was allowed to sit
`at ambient temperature for 24 h. The dispersion was centrifuged to
`pellet the solids. The supernatant was filtered through a 0.22-pm
`Millipore Millex-GV filter and assayed by HPLC for insulinotropin
`concentration.
`Analytical HPLC assay of insulinotropin was conducted on a Vydac
`Protein C4 column (Rainin Cat# 214TP54). The gradient program used
`for the assay is listed below
`
`time (min)
`Yo A
`%B
`
`0
`75
`25
`
`5
`75
`25
`
`30
`50
`50
`
`35
`50
`50
`
`37
`75
`25
`
`46
`75
`25
`
`where A represents 0.1% trifluoroacetic acid (TFA) in water and B
`represents 0.1 % TFA in acetonitrile. The flow rate was set at 1 mL/min
`
`0 1994, American Chemical Society and
`American Pharmaceutical Association
`
`0022-3549/94/1200- 1 175$04.50/0
`
`Journal of Pharmaceutical Sciences / 1 I75
`Vol. 83, No. 8, August 1994
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`Table 1-Equlllbrlum Solublllty of Samples A-D In pH 7.1
`Phosphate-Buffered Saline
`Samples
`Solubility (mg/mL)
`1
`A
`0.001
`B
`
`Samples
`c
`D
`
`Solubility (mg/mL)
`0.47
`0.001
`
`and the column kept at ambient temperature (25 "C). Detection was
`by UV at 215 nm. Insulinotropin retention time was ca. 23 min.
`The equipment used for the HPLC assay consisted of an LDC Consta-
`Metric 4100 solvent delivery system, a Bio-Rad Model AS-100 HRLC
`automatic sampling system, an LDC SpectroMonitor 4100 programmable
`variable-wavelength detector, and a Spectra-Physics Chrom Jet integra-
`tor.
`Vibrational Spectroscopy-FT-IR spectra of proteins were obtained
`by microinfrared measurements, using an IR-MAU100 unit with a MCT
`detector (IR-DETlO2) attached to a JEOL JIR-6500 FT-IR spectrom-
`eter.'* All the spectra were recorded at a 4-cm-l resolution with 200-
`1000 scans. The proteins were put on a Ge window and crushed to
`obtain satisfactory spectra. The second-derivative spectralg were
`calculated (smoothing points, 13) by using JEOL JIR-6500 FT-IR
`software. The percentage contributions of the a- and @-conformations
`were calculated on the basis of the method of Dong et a1.20 All these
`calculations have been made using the area under the peaks from the
`second-derivative spectrum.
`Near-IR FT-Raman spectra of proteins were measured at an 8-cm-1
`resolution with a JEOL JRS-FT 6500N Raman spectrometer equipped
`with an InGaAs detector.2I The 1064-nm line of a CW NdYAG laser
`(Spectron SL301) was used for excitation, and the laser power employed
`at the sample position was 350-850 mW. Raman scattering light was
`collected with a 180" backscattering geometry, and all the spectra were
`the result of the coaddition of 2000-4000 interferograms.
`Hydrophobicity Analysis-Hydrophobicity analysis of this peptide
`was possible since the primary sequence was known. The Kyte and
`Doolittle scale of hydropathy was used to evaluate the hydrophilic and
`hydrophobic regions of the peptide.22 This method uses a moving-
`segment approach that continuously determines the hydropathy within
`a segment of predetermined length (n) as it advances through the
`sequence from the amino to the carboxy terminus.22 Six is often chosen
`as n, so that the segment length is larger than the oscillations associated
`with the periodic helix or strand and yet sufficient local information is
`retained. For insulinotropin (31-amino acid peptide), an average of
`hydropathy over six residues was calculated up to the 26th residue, and
`then the n was reduced by one each time as the C-terminal was
`approached.
`spectrum was
`Circular Dichroism (CD) Measurements-CD
`recorded on a 5-720 Jasco circular dichroic spectropolarimeter in 0.01-
`cm pathlength cells at 25 "C. The peptide concentration was 0.8 mM
`in a 50 mM phosphate buffer at pH 8.
`Water Content Determination-Water contents in thesolid samples
`were determined by two different methods: coulometric Karl Fisher
`titration23 (Mitsubishi moisture meter, Model CA-06, Mitsubishi Kasei
`Corp., Tokyo, Japan) and thermogravimetric analysis" (TGS-2 analyzer,
`Perkin-Elmer, Norwalk, CT).
`
`Results
`Solubility Behavior-The equilibrium solubility in PBS for
`samples A-D are summarized in Table 1. The solubility of the
`original synthetic insulinotropin (sample A) is 1 mg/mL. If an
`insulinotropin solution in water or PBS is stirred vigorously,
`highly insoluble aggregates are formed. The isolated solids
`(sample B) have a solubility of about 1 pg/mL in PBS. The
`solubility of the phenol precipitate of insulinotropin (sample C)
`is 0.47 mg/mL in PBS. Sample D constitutes the aggregates
`isolated from an insulinotropin solution and a phenol solution
`that were combined and then stirred overnight. This sample
`has a solubility of about 1 pg/mL in PBS.
`Peptide Backbone Analysis by FT-IR-The band assign-
`ments for different conformations are studied by many inves-
`tigators are summarized in Table 2.Z5s26
`
`1176 / Journal of Pharmaceutical Sciences
`Vol. 83, No. 8, August 1994
`
`Table 2-IR Band Assignment of Amlde I and I1 to Dtfferent
`Conformers of Polypeptide Studled by Other Investlgators2s~2a
`Frequency (cm-l)
`Amide I
`Amide 11
`1650- 1655
`15 16-1 546
`1632- 1635
`1530
`1668 (theoretical)
`1685
`1630
`1645
`1685- 1690
`1660
`
`~
`
`1530-1550
`
`Not studied
`Not studied
`
`Conformer
`
`a-Helix
`Antiparallel P-sheet
`
`Parallel @-sheet
`
`@-Turn
`10 Helix or distorted a-helix
`
`WAVENUMBER CM'
`
`-0.0080
`
`1654
`
`FT-IR spectrum of synthetic insulinotropin (sample A). (B)
`Figure 2-(A)
`Second-derivative of sample A.
`
`The FT-IR spectra of the solid insulinotropin samples (A, B,
`C, and D) made under different conditions are shown in Figures
`2A, 3A, 4A, and 5A, respectively, and their frequencies in the
`amide I and 11 regions are summarized in Table 3.
`Instead of relying only on the original FT-IR spectra, the
`second-derivative spectrum of each sample was also obtained
`(Figures ZB, 3B, 4B, and 5B, respectively) since small shoulders
`in the spectrum can be seen as clear peaks in the second-derivative
`spectrum. The area under these peaks can be used to determine
`
`the percentage composition of the various stru~tures.~g~~O The
`contents of different conformers for samples A-D were obtained
`by analyzing Figures ZB, 3B, 4B, and 5B, and are summarized
`in Table 4. The differences in the amide regions of the spectra
`of the four samples are very clear and distinct. The peak at 1654
`cm-1 is strong and distinct only in the spectrum of sample A.
`Sample B shows an increase in intensity of the peak at 1668 and
`1635 cm-l. Sample C has a very intense peak at 1635 cm-l. Sample
`D, on the other hand, has two very intense peaks at 1668 and
`at 1632 cm-'. These clear differences can allow us to draw valid
`conclusions on structural differences among the four samples.
`For sample A, a major peak was observed at 1657 cm-1 (Figure
`2A). This is an indication of an a-helix.
`The corresponding second-derivative spectrum reveals much
`more information (Figure 2B). The main peak is at 1654 cm-1,
`which is an indication of an a-helix (49%). The 1685-cm-1 band,
`shown in the second-derivative spectrum as a small but distinct
`
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`A
`
`0.2000
`
`0.1500
`
`a
`
`c : 0.100c
`0 3
`
`0.0500
`
`0. 0000
`41
`
`3280 i' \,
`
`j002
`
`Figure 3-(A) FT-IR spectrum of sample B. (B) Secondderivative spectrum
`of sample 8.
`
`0.4000-
`
`A
`
`I 610
`
`I6607
`
`FT-IR spectrum of sample D.
`Figure 5-(A)
`spectrum of sample D.
`Table 3-Frequency Observed for Insullnotropln Samples A-D
`In Amide I and I1 Regions Studied by FT-IR
`Frequency (cm-')
`Amide I1
`Amide I
`1537
`1657
`1635 (major)
`1543
`1668 (minor)
`1630 (major)
`1660 (minor)
`1630 (major)
`1666 (minor)
`
`(B) Secondderivative
`
`1545
`
`1539
`
`Sample
`A
`B
`
`C
`
`D
`
`A h
`
`backbone conformation when insulinotropin is subjected to high
`shear force.
`Sample C is the precipitate formed when an insulinotropin
`solution is combined with phenol solution. The IR spectrum of
`sample C is basically similar to that of sample B with peaks at
`1630 and 1660 cm-I (Figure 4A). The presence of these
`antiparallel &sheet bands is also seen in the second-derivative
`spectrum (Figure 4B). However, in contrast to sample B, a
`Y ,I
`shoulder was observed at 1655 cm-I (Figure 4B). This is an
`1600
`1640
`indication of an a-helix structure, which was quantitated to be
`WAVENUMBER CM'
`FT-IR spectrum of sample C.
`Figure 4-(A)
`21 ?6 of the overall structure.
`spectrum of sample C.
`Sample D was prepared by applying shear force to sample C
`before it was isolated from the mother liquor. The FT-IR
`spectrum of sample D (Figure 5A) is very much like those of
`samples B and C. However, the second-derivative spectrum
`(Figure 5B) is more similar to that of sample B (Figure 3B) than
`sample C (Figure 4B). For sample B, there are two bands in the
`1632-cm-l region, instead of one band as for sample D.
`There are broad bands in the region of 2800-3100 cm-1 in the
`spectra of all samples (Figure 2-5). These bands are constituted
`of C-H stretching vibration, overtones of C-H bending, and
`Fermi resonance bands. The interpretation of these bands would
`be very important, if the samples were lipids or membranes.
`However, no correlation was reported between the secondary
`and tertiary structures of proteins and these bands. Therefore,
`no further analysis was carried out on these bands. The distinct
`
`I
`
`-0.0080
`
`-0.0120 I
`I300
`
`1760
`
`1720
`
`1680
`
`1560
`
`1520
`
`1480
`
`1440
`
`1400
`
`(B) Secondderivative
`
`peak, indicates the presence of a small amount of an antiparallel
`&sheet or &turn.
`Sample B shows dramatic changes in its FT-IR and second-
`derivative spectra as compared to sample A. The a-helix band
`at 1657 cm-1 is no longer visible (0% a-helix). Moreover, the
`amide I band has two peaks at 1635 cm-' (major) and at 1668
`cm-1 (minor) in the FT-IR spectrum (Figure 3A). The second
`derivative spectrum (Figure 3B) also clearly indicates the
`presence of bands at 1635 and 1668 crn-'. Both of the 1635 and
`1668 cm-l bands are indications of an antiparallel p-sheet
`structure. Asmall band at 1645 cm-l is observed in the derivative
`spectrum, but not in the original IR spectrum. The spectra of
`Figure 3A,B clearly signify that there is a dramatic change in the
`
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`Table 4-Characterlstlc Band Frequencles and the Content of
`Dlfferent Conformers for Insulinotropin Solids Prepared by
`Different Methods
`Sample Frequency (cm-') Conformation Assignment Content (%)
`1654 (very large)
`a-Helix
`A
`49
`Antiparallel /.?-sheet
`1635
`13
`1685 (small)
`Antiparallel @-sheet
`12
`Random coila
`26
`Antiparallel &sheet
`22
`Antiparallel &sheet
`32
`Random coila
`46
`Antiparallel @-sheet or
`49
`parallel &sheet
`a-Helix
`Distorted a-helix or
`antiparallel &sheet
`Random coila
`Antiparallel @-sheet or
`parallel @-sheet
`Antiparallel @-sheet
`27
`Random coila
`34
`aThe content of random coil is calculated from the difference
`between the total content and the summation of a-helix and ,&sheet
`structure contents.
`
`B
`
`C
`
`1635 (large)
`1668 (very large)
`
`1630 (very large)
`
`1655 (shoulder)
`1666 (broad and
`moderate size)
`
`D
`
`1632
`
`1666
`
`21
`37
`
`0
`39
`
`~
`
`peak at 3280-3290 cm-1 is obviously from OH vibration of the
`water molecules present in the sample and the NH vibration of
`the amino groups of the peptide. In all of these samples the
`amide I1 band is very distinct (1539-1545 cm-I). This amide I1
`band is Raman inactive and thus is not seen in the Raman spectra
`(Figure 6).
`spec-
`Near-Infrared FT-Raman Spectroscopy-Raman
`troscopy has been employed extensively for protein conformation
`identification for the last 2 decades. Recently near-infrared FT-
`Raman spectroscopy has been rapidly developed, as it has
`advantages over conventional Raman spectroscopy. Near-
`infrared excitation can avoid fluorescence background and largely
`reduce photodecomposition. Therefore, FT-Raman spectros-
`copy provides Raman spectra with highly accurate frequencies
`with clean background^.^^ For the present study, the amide I
`and I11 regions were examined carefully.28-30
`For the a-helix conformation, the amide I frequency is
`relatively low, ranging from 1642 to 1658 cm-l. The antiparallel
`@-sheet structure is in the higher frequency range of 1662-1680
`cm-l. Frequencies due to random coil structures are between
`the frequencies of the a-helix and @-sheet, i.e., between 1660 and
`1665 cm-1. The amide I11 band of the a-helix appears at 1260-
`1300 cm-1, while that of the random coil has relatively low
`frequencies of 1240-1250 cm-I. For the /3-sheet structure, the
`amide I11 absorbs at 1242-1260 cm-l.
`The original synthetic insulinotropin (sample A) gives a
`distinct amide band at 1659 cm-l, indicating the presence of an
`a-helical conformation (Figure 6A). Although the main peak is
`located at 1659 cm-l, there are shoulders at 1685 and 1695 cm-1.
`This suggests that the secondary structure of sample A consists
`mainly of an a-helix, but some amount of &sheet is also present
`in the native, untreated insulinotropin.
`For the amide I11 region, there are continuous ascending bands
`from 1250 to 1300 cm-1. This region is a highly mixed vibrational
`zone. For an accurate determination of amide I11 bands, it is
`usually recommended to dissolve the protein of interest in D20
`to allow isotopic exchange. The deuterated protein should give
`a new amide 111' band in the vicinity of 980 cm-1, and any bands
`not shifted in the region of 1200-1300 cm-l are not amide I11
`bands.28~29 Isotopic exchange of hydrogen and deuterium atoms
`induced by dissolving sample A in D20 caused these bands to
`disappear and shift to lower frequencies (the spectrum is not
`
`1178 / Journal of Pharmaceutical Sciences
`Vol. 83, No. 8, August 1994
`
`1003
`
`1335
`
`1.2000
`
`0.8000
`
`1659 I
`
`0.4000
`
`0.0000
`
`I666
`I
`
`1870
`
`B
`
`I003
`
`I h
`
`D
`
`0,3200
`
`>
`I- o.2800
`a z
`W
`i- z
`Z a
`2 0.2000
`a
`U
`
`0.2400
`
`0,2400
`
`0,1600
`
`0.1200
`
`0.4000
`
`0.3200
`
`0,2400
`
`0.1600
`
`1700
`
`1600
`
`1500
`
`1300
`I200
`I100
`1400
`WAVENUMBER CM'
`Flgure 6-FT-Raman spectra of insulinotropin in the solid state: (A) sample
`A, (B) sample B, (C) sample C, (D) sample D.
`
`1000
`
`900
`
`BOO
`
`shown). This suggests that the continuous ascending bands in
`1250-1300 cm-' are an expression of an a-helix.
`Due to the low solubility of samples B-D, hydrogen/deuterium
`isotopic exchange could not be carried out. The Raman spectra
`of samples B-D are shown in Figure 6B-D.
`The amide [ bands of sample B-D are quite similar to each
`other: 1666 cm-' for sample B, 1666 cm-l for sample C, and 1670
`cm-' for sample D. This is an indication of an antiparallel &sheet
`structure for these samples. Resolution of the amide I band for
`Raman is not as good as that for FT-IR, but it clearly shows that
`there is a shift of a-helix to 8-sheet structure from sample A to
`sample B-D. The decrease in solubility (Table 1) from sample
`A to samples B-D can be correlated with a major conformation
`change from a-helix to 0-sheet structures.
`The C-H bending vibrational mode is strongly Raman active,
`and an intense Raman band is observed at 1450 cm-1 in all
`samples. However, this band is very weak in the IR spectra
`(Figures 2-5)
`Hydrophobicity Analysis-The hydrophobicity analysis of
`insulinotropin (Figure 7) indicated that the region Ala(lS)-Ala-
`(24) is the most hydrophobic. This hydrophobic region is located
`between two hydrophilic regions, Ser(ll)-Gln(l7) and Trp(25)-
`Gly(31). The His(l)-Val(lO) is neither very hydrophobic nor
`very hydrophilic. These regions would therefore determine the
`
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`3
`
`2
`
`,
`
`0
`
`
`
`t
`
`0
`
`0
`
`a e
`
`r
`
`”
`
`l
`
`
`
`2
`
`1 2 3 4
`
`5 6
`
`7 8 9 10 1 1 12 13 I 4 15 16 17 18 19 70
`
`22 23 24 25 26 77 78 29 30 1 1
`
`Residue number
`Figure 7-Hydrophobicity of insulinotropin. The values were calculated
`by the Kyte and Doolittle method.**
`
`i IOEiOl
`
`1 WE+U
`
`I SOEtOl
`
`190
`
`ZM
`
`210
`
`x n
`ZIO
`Wwdcnpb (nm)
`
`Z*O
`
`zso
`
`1M
`
`Figure 8-Far-UV CD spectrum of insulinotropin in solution. The peptide
`concentration was 0.8 mM in a 50 mM phosphate buffer at pH 8.
`
`manner in which the peptide would fold under different
`environmental conditions such as solvent, pH, and ionic strength.
`Circular Dichroism-Figure 8 shows the far-UV CD spec-
`trum of insulinotropin in solution. The spectrum is characteristic
`of an a-helical conformation: two minima at 207 and 223 nm
`and a maximum at near 190 nm.31
`Water Content Determination-Sample A was the only dry
`powder, and samples B-D were wet cakes. Water content
`determinations were performed in duplicate for each sample.
`The two methods, thermogravimetric analysis and coulometric
`Karl Fisher titration, showed almost the same results, although
`the former tended to give a little higher (0.3% higher on an
`average) water content values than the latter. The water content
`of sample A was 6.8% w/w, and those of the wet samples (B-D)
`were in the range of 85-87% w/w.
`
`Discussion
`Insulinotropin is composed of 31 amino acid residues. The
`hydrophobicity profile indicates that the highest hydrophobicity
`region lies between Ala(18) and Ala(24). The original synthetic
`insulinotropin (sample A) has a high solubility in aqueous media
`with a high a-helix content, as demonstrated in the CD spectrum
`(Figure 8). It suggests that the Ala(18)--Ala(24) hydrophobic
`region must be buried inside by the molecule and the hydrophilic
`regions must be in the exposed state. We can presume that the
`two hydrophylic regions, Ser(l1)-Gln(l7) and Trp(25)-Gly(31),
`form a-helixes and are in good contact with water molecules.
`Prestrelski et al. reported dehydration-induced conformational
`changes in proteins.32 They observed conformational transitions
`
`from a helical conformation or an unordered structure to a P-sheet
`after freeze-drying protein solutions. Similarly, we observed an
`a-helix to @-sheet transition when the original peptide (sample
`A) in solution was exposed to phenol (sample C) or high shear
`force (samples B and D). Samples A was the only dry powder
`(moisture content = 6.8% w/w), and the other samples (B-D)
`were not subjected to any kind of drying process besides
`centrifugation (moisture contents 2 85% w/w). Therefore, it is
`difficult to relate the cause of the a to @ conversion observed in
`our study to dehydration.
`Ismail et al. demonstrated that certain denaturing conditions
`could cause irreversible intermolecular self-association of a
`peptide that was accompanied by the appearance of a very strong
`IR absorption band at 1618 cm-l.33 Since we have not observed
`such a band in the 1610-1620 cm-l region of the IR spectra for
`any of the insulinotropin samples, it is unlikely that the decreased
`solubility for the precipitated samples is attributed to irreversible
`self-association. The high-shear-induced precipitates (samples
`B and D), which have the lowest solubility in neutral pH range
`(1 jtg/mL solubility in PBS or water), are in fact readily soluble
`in an acidic solvent such as 0.01 N HC1 or 0.1 % trifluoroacetic
`acid in water. We have performed an in vitro bioactivity test34
`with the high-shear-induced precipitates of insulinotropin after
`redissolving them in an acidic solvent. No differences in
`bioactivity were observed between the original peptide (sample
`A) and the precipitated samples, and all of them were fully active.
`The self-association of synthetic insulinotropin in solution
`was characterized by Grucza et al. using equilibrium analytical
`ultracentrifugation and CD spectroscopy.35 It was determined
`that the peptide formed a tetrameric species with significantly
`higher a-helical content than the monomer. They also observed
`that the tetramers precipitated out of the solution upon storage
`at ambient temperature overnight, but the aggregation was
`completely reversible.
`It has been suggested that proteins in solution are destabilized
`by protein adsorption at hydrophobic interfaces (air-water
`interface or water-container materials), and that the initial step
`is nucleation. Partially or completely unfolded protein molecules
`form nuclei which serve as precursors to large aggregates.-O
`Since insulinotropin does not contain any disulfide bonds, the
`peptide may not have a tightly packed three-dimensional
`structure. With shaking or stirring, the air-liquid and the liquid-
`solid interfaces increase, leading to greater conformational
`changes and denaturation. From the results in Table 4, it is
`evident that insulinotropin underwent conformational changes
`when it was subjected to vigorous agitation. For the samples B
`and D, the contents of the @-sheet and random coil increased at
`the expense of the a-helix. The mechanism of this conforma-
`tional conversion is not understood, however. This may not be
`a single event.
`The helix to random coil transition is one of the most
`thoroughly studied phenomena. Interconversion of the two
`structures can occur upon changing the pH, the solvent, the
`temperature, etc., and the transition is very abrupt, indicative
`of a cooperative system.41 However, the mechanism of @-sheet
`formation is not well understood. Synthetic polypeptides that
`form &sheets generally produce large, insoluble structures
`comprising many peptide chains.41 With the limited information
`available, one can propose two separate events occurring.42 At
`first, insulinotropin molecules lose their helical structure and
`become disordered due to the hydrophobic interfaces. Then,
`the disordered chains aggregate, forming a &sheet structure,
`which results in a low solubility in aqueous media. A kinetic
`study is being carried out in our laboratory to elucidate the
`mechanism of this phenomenon.
`When insulinotropin is mixed with phenol without stirring
`(sample C), the solubility is still low (470 jtglmL), but much
`higher than that of samples B and D, which were stirred (1 pg/
`mL). Both IR and Raman spectroscopy provide evidence for
`
`Journal of Pharmaceutical Sciences / 1179
`Vol. 83, No. 8, August 1994
`
`Novo Nordisk Ex. 2035, P. 5
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`
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`42.
`
`Acknowledgments
`We acknowledge the advice given by Dr. Wayne A. Boettner for the
`preparation of this manuscript. We thank Dr. Cheryl M. Kirkman for
`the HPLC method development of insulinotropin and Mr. Geoffrey
`Stamper for the CD measurement.
`
`the significant decrease in the a-helix content in sample C with
`respect to sample A. This sensitivity of the helix to phenol may
`indicate that a certain component, such as a specific amino acid
`residue that is important in the maintenance of the helical
`structure, can be easily disrupted by phenol, possibly by the OH
`group.
`In this study of insulinotropin, the relationship between
`solubility and peptide backbone conformation is demonstrated
`by vibrational spectroscopic evidence. Specifically, the decrease
`in a-helix content is definitely related to the decrease in solubility
`of insulinotropin.
`
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