Biochimica et Biophysica Acta 1814 (2011) 318–325

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FTIR 2D correlation spectroscopy of α1 and α2 fractions of an alkali-pretreated gelatin

Pieter Chys a, Constant Gielens a, Filip Meersman b,⁎a Katholieke Universiteit Leuven, Department of Chemistry, Division Biochemistry, Molecular and Structural Biology, Celestijnenlaan 200 G, 3001 Leuven, Belgiumb Division Molecular and Nanomaterials, Celestijnenlaan 200 F, 3001 Leuven, Belgium

⁎ Corresponding author. Tel.: +32 16327355; fax: +E-mail addresses: Pieter.chys@chem.kuleuven.be (P.

Constant.Gielens@chem.kuleuven.be (C. Gielens), Filip.M(F. Meersman).

1570-9639/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.bbapap.2010.10.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 February 2010Received in revised form 24 September 2010Accepted 4 October 2010Available online 13 October 2010

Keywords:Gelatinα fractionFTIR spectroscopyAlcohol coacervationFractionationSol–gel transition2D COS

An alkali-pretreated gelatin (pI~4.9) was fractionated bymeans of alcohol coacervation and semi-preparativegel chromatography. The thermal responses of the isolated α fractions, the coacervate and the total gelatinwere investigated by 2D-correlation FTIR spectroscopy in the amide I band region (1600–1700 cm−1). Thegelation temperature was the same for all examined samples (24.5 °C) while the melting temperature of theα2 fraction was lower (30 °C) than that of the other samples (32.5 °C). The 2D COS plots indicate that oncooling (gelation) the core sequence of conformational changes is the same for all samples. On heating,however, the α2 fraction deviates from the α1-containing samples and shows an earlier disappearance of thetriple helix signal in the event sequence. The lower melting temperature (less thermostable gelatin gel) of theα2 fraction thus results from a different conformational cascade of the α2 chains upon melting. In all samplesthe initial conformational changes take place in the β-turns, providing further evidence for the modelsproposed previously.

32 16327990.Chys),eersman@chem.kuleuven.be

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Gelatins are denatured collagen molecules with a wide range ofapplications [1,2]. The collagen molecule is one of the most importantstructural proteins in the animal kingdom [3]. A variety of gelatinsexists originating from different collagen types, manufacturingprotocols, Mr distributions and so on [4,5]. From the economic pointof view, gelatins account for a substantial amount of the biomaterialtraded in the food industry [6].

The physicochemical nature of gelatins is unique [4,5]. Gelatinsbehave completely different in solution as compared to globularproteins and are random-coil like and extended instead of native andcompact. A high degree of monodispersity is difficult to attain formanufactured gelatins and most gelatin samples cover quite a broadMr range [4,5,7], which is both a strength and drawback. Dependingon solute (concentration c, type) and solvent conditions (e.g. pH, T,alcohol content, salt c, salt type, etc.) a whole range of states can exist,including diluted sol, coacervated system, aqueous gel. In addition,gelatins are weak polyampholytes, the behavior of which is highlydependent on the pH of the solution [8–13]. The above features resultin the ability of gelatin solutions to form physical gels when thegelatin concentration is high enough [4,5]. Such gelatin gels exhibit aviscoelastic sol–gel transition and are thermoreversible in nature.However, gelation (Tg) and melting (Tm) temperatures for the same

gelatin gel mostly differ and result in temperature hysteresis ofexperimental cooling (gelation) and heating (melting) curves,indicating a different stability upon heating and cooling of the gelatingel [4].

The looseness and randomness of the gelatin architecture insolution is in sharp contrast to the native structure of collagen: a stifftriple helix (tropocollagen) of three α chains [5,14]. The constituting αchains (Mr~95 000) remain the building blocks for gelatins but allgeometric constraints on conformation, such as present in tropocol-lagen, are lost in solution. In addition, the α chains are not always thesame in a single tropocollagen molecule of a given biological species[3,15]. At present, approximately 30 types of tropocollagen are knownof which some consist of a single and unique α chain, whereas othersdo not [16]. For example, in collagen type I, the main source forgelatins in industry, two α1 chains and one α2 chain, build up thetropocollagen macromolecule.

The purpose of the study here is to examine in detail the thermalbehavior of α1 and α2 fractions and to investigate whether or notthese α fractions behave differently. To this end α1 and α2 chains froma total alkali-pretreated gelatin were fractionated by means ofmethanol coacervation and semi-preparative gel chromatography.The fractions were subsequently characterized by one- and two-dimensional Fourier transform infrared (FTIR) correlation spectros-copy (2D COS) during a cooling–heating cycle (50 °C→5 °C→ 50 °C).

2. Materials and methods

An alkali-pretreated gelatin (gelatin PB 88210) was kindlyprovided by PB Gelatins, Tessenderlo Chemie (Vilvoorde, Belgium).

319P. Chys et al. / Biochimica et Biophysica Acta 1814 (2011) 318–325

It is a demineralised limed gelatin from cattle bones (single batch).The Bloom value is 285 g and the isoelectric point is ~4.9.

Gelatin PB 88210 is highly polydisperse and consists of fivefractions [17]. The principal fraction is the α fraction which has twosubfractions [5], α1 and α2, which originate from the α chainsconstituting collagen type I. The β and γ fractions are double andtriple covalently cross-linked α chains, respectively. Microgels(MrN N106) are built up of more than three cross-linked α chains.The fifth fraction consists of α chain fragments.

2.1. Fractionation of total gelatin

To obtain enriched α1 and α2 fractions we used a combination ofsimple alcohol coacervation and semi-preparative gel chromatography.

2.1.1. CoacervationThe coacervation protocol is modified from Veis [4]. Gelatin grains

were added to an aqueous 0.8 M NaCl solution at 2% (w/v) and keptfor 1.5 h at room temperature (22 °C) to allow for swelling. Thismixture was then heated at 50 °C for 2 h [17,18]. Next, hot methanol(40–50 °C) was slowly added (5 min) in a 2.5/1 v/v ratio to the warmgelatin solution. After homogenization the mixture was allowed tocool and kept overnight at room temperature (15–16 h). Thesupernatant, consisting of the α2 fraction, was decanted andlyophilized at –30 °C to remove the methanol. The dry product wasredissolved in ultrapure water, dialyzed against ultrapure water andlyophilized again. The coacervate was appropriately diluted to obtaina solution and heated after which it was also lyophilized.

2.1.2. Gel chromatographyTo obtain an enriched α1 fraction, the coacervate (7.4 mL at a

concentration of 2.6% (w/v)) was applied on a Sephacryl S-400 HRcolumn (86 cm×4.0 cm) and eluted. The column was thermostattedat 40 °C to prevent gel formation during elution of the applied sample.Eluent composition was 50 mM ammonium acetate (pH~6.9), 0.02%(w/v) sodium azide. Flowwas 0.8 mL/min and separation range of thecolumn is 2×104−8×106 Da for globular proteins (1×104

−2×106 Da for linear dextrans). After location of the α1 chains inthe eluate, selected tube fractions were collected and lyophilized.

Gelatin samples were identified by means of Fast Protein LiquidChromatography (FPLC) on a Superose 6 HR 10/35 column (Amer-sham Biosciences). The column has a separation range for globularproteins from 5×103 to 5×106 Da (exclusion limit ~4×107 Da). Flowwas 0.2 mL/min at room temperature and the eluent was 80% (v/v)50 mM sodium phosphate buffer, pH 6.9, 20% (v/v) n-propanol and0.15 M NaCl. Working pressure was 0.8–0.9 MPa. FPLC was also usedto determine the composition of the total gelatin sample for reference.This sample was made as described above but without thecoacervation step. For all samples, after spectrophotometric determi-nation of gelatin concentration (a230=2.0 L/g·cm [17]), an appropri-ate dilution [0.3% (w/v)] was made and the samples were heated for1 h at 50 °C. A volume of 300–600 μL of the warm solution wasinjected.

2.1.3. ElectrophoresisSodium dodecyl sulphate electrophoresis (SDS-PAGE) allows the

discrimination between gelatin α1 and α2 fractions [19]. Theelectrophoresis was performed on an LKB 2050 Midget apparatususing a current of 25 mA per gel. Stacking gel was 4% (w/v)polyacrylamide and running gel 7.5% (w/v) polyacrylamide. Electro-phoresis buffer was Tris (25 mM)–glycine (190 mM), 0.1% (w/v) SDS,pH 8.9. Generally 20 μL samples were applied to each lane of the gel.An amount of 5 to 10 μg of gelatin per sample is needed. If the solutionvolumewas larger than 10 μL, the samplewas first driedwith a speed-vac concentrator (SC 110 AR, Savant). To the sample, 20 μL samplebuffer, containing 2% (w/v) SDS and 1% (v/v) 2-mercaptoethanol, was

added. This mixture was heated at 100 °C for 3–4 min. After theelectrophoresis, the gel was carefully removed from the glass platesand placed for 30 min in a coloring solution of Coomassie BrilliantBlue R250 [0.25% (w/v) in methanol/acetic acid/water 5/1/5 (v/v/v)].After this, the gel was destained in a decoloring solution [methanol/acetic acid/water 2/3/35 (v/v/v)].

2.2. FTIR spectroscopy

Samples of α1, α2, coacervate and total gelatin in D2O [5.0% (w/v)]were subjected to a thermal cycle: warm gelatin sols of 50 °C werecooled down to 5 °C and upon reaching 5 °C, the samples wereimmediately reheated to 50 °C. Cooling and heating rate was 0.2 °C/min.

One-dimensional spectra [20,21] were obtained with a BrukerIFS66 Spectrometer equipped with a liquid nitrogen cooled mercury-cadmium-telluride (MCT) detector. The sample compartment wascontinuously purged with dry air. At each temperature, 256interferograms (resolution 2 cm−1) were averaged for each spectrum.The temperature cell is made up by two CaF2 windows and a 50 μmteflon spacer, and is controlled by an Automatic TemperatureController of Graseby Specac.

Two-dimensional correlation spectroscopy (2D COS) of the one-dimensional FTIR spectra was implemented in MATLAB [22]. A singlewindow over the entire temperature range of the experiment(5 °C−50 °C) was used. From the complete series of FTIR spectra asa function of temperature, the raw data of the amide I band weretaken, smoothed twice by spline interpolation and a linear back-ground (1590–1700 cm−1) subtracted. To check the validity of thebaseline correction a wider wavenumber range (1400−1900 cm−1)was tested in 2D COS calculations but this made no difference andtherefore the narrow range was used. Next, the dynamic componentof the FTIR spectra was calculated by subtraction of the staticcomponent. Since subtraction of an average static spectrum canamplify noise and introduce false peaks when the dynamic compo-nent has relatively low amplitude versus the static component [23],we used the steady-state spectrum as reference. For cooling this wasthe 50 °C spectrum, for heating the 5 °C spectrum. Since 2D COS plotswith no scaling yielded good results, we kept the computationalprocedure as simple as possible and avoided scaling techniques. Noscaling was thus done after computation of the dynamic component.The discrete Fourier transform and its conjugate transform for a givenstructural event pair (e.g. change at ν1 versus change at ν2) could nowbe calculated and separately summed. A complex number for eachpossible pair of wavenumbers (ν1,ν2) is obtained: Φ+Ψi. The realcomponent Φ of this number represents the synchronous componentof the correlation, the imaginary partΨ the asynchronous component.From the real and imaginary part of Φ+Ψi, synchronous andasynchronous plots are constructed and interpreted according to therules of Noda [24]. We introduce a graphical representation whichshows the information of both separate component plots in one, withthe synchronous information in the lower triangle and the asynchro-nous in the upper triangle. Construction of such a graph is as follows.The lower triangle of the synchronous plot is first plotted at its normalplace on an empty 2D COS grid. Hereafter, the lower triangle of theasynchronous plot is orthogonally reflected along its main diagonaland plotted into the upper triangular area. Thus, the Y axis of the new2D COS plot corresponds to the original X axis of the asynchronousplot in case of the asynchronous (upper) triangle. Since both the lowertriangles of the component plots are used, the rules of Noda [24]remain valid. This type of graph has the advantage that the dataredundancy in both separate plots is eliminated. Since the asynchro-nous component was much smaller in general, we scaled theasynchronous component data range towards the range of thesynchronous component for uniformity of the color scale. For boththe synchronous and asynchronous triangles, cross-peaks are referred

Fig. 2. SDS-PAGE of total gelatin and fractions obtained by methanol coacervation ofgelatin (volume ratio methanol/gelatin sol: 2.5/1).

320 P. Chys et al. / Biochimica et Biophysica Acta 1814 (2011) 318–325

to by using X/Y cm−1 [− ,s], with X and Y the associated wavenumberpositions at the horizontal and vertical axes of the 2D COS plot. Thus,1657/1623 cm−1 refers to the cross-peak in the synchronous trianglewhereas 1623/1657 cm−1 refers to its counterpeak in the asynchro-nous triangle. Additional information in between brackets is given forthe sign (+ or−) and relative magnitude (s=strong, w=weak or s/w=medium) of the peak.

3. Results

3.1. Isolation of α1 and α2 fractions

Fig. 1 shows the FPLC chromatogram of the total gelatin (solidblue) as well as a Gaussian polyfit for 6 fractions. The fragments weredivided in two fractions thereby yielding a sixth fraction. The αfraction (red dashed) at 11.5 mL is the most important fraction,consisting of both the α1 and α2 fractions, which have an identicalmolecular mass.

The SDS-PAGE from total gelatin, supernatant and coacervate afterthe methanol coacervation is shown in Fig. 2. In electrophoresis lane 1(total gelatin) two bands are observed near the α positioncorresponding to the two α fractions. Electrophoresis results inAoyagi et al. [19] show the upper band to be α1 and the lower α2. TheSDS-PAGE reveals that α1 enters mainly the coacervate whereas α2

remains mostly in the supernatant. This electrophoresis demonstratesthat the two α fractions are largely separated by the methanolcoacervation. Thus, the supernatant of an appropriate coacervationyields an enriched α2 fraction and the coacervate contains mostly α1.The coacervate can be further used to enrich the α1 fraction.

The elution profile on Sephacryl S-400 HR of the coacervate fromthe methanol coacervation (Fig. 3) shows a broad band and no cleardistinct peaks for the different fractions of the coacervate. However,samples taken over the complete elution range (black and numberedticks in Fig. 3) and subjected to FPLC show that the selected fractionshave different compositions (Fig. 4). Tube fraction 130 can beidentified as corresponding to an enriched α fraction (see Fig. 1)and, since the coacervate is mainly devoid of α2 this fractionconstitutes enriched α1 fraction.

3.2. FTIR spectroscopy

FTIR spectroscopy was used to determine the gelation (Tg) andmelting (Tm) temperatures, and mechanistic insight into the sol–geltransition was obtained by a 2D COS analysis. The changes in

Ve (mL)

A a

t 214

nm

6 8 10 12 14 16 180

0.1

0.2

0.3

0.4

0.5

0.6

γβ

α

total

fragments 1

fragments 2

micro−gels

Fig. 1. Composition of the total alkali-pretreated gelatin (solid blue) and a Gaussianpolyfit. The solid blue curve (total) was experimentally determined by FPLC.

secondary structure were monitored through the conformationallysensitive amide I band that results from the carbonyl groups inproteins and is strongly influenced by the local surrounding anddipole coupling to other carbonyl groups [20,21]. Table 1 shows, nextto the empirical secondary structure–wavenumber correlations foundfor globular proteins [25–27], also assignments proposed for gelatin[28] and poly-L-proline [29].

3.2.1. Gelation and melting pointFig. 5 shows the thermal response of the amide I band for a total

gelatin sample during cooling. Around 1657 cm−1 the peak increasesupon cooling, indicating the formation of triple helix structure inagreement with previous observations [28,30]. Upon sufficient cool-ing and subsequent gelation an infinite gelatin network forms and it isgenerally assumed that partially refolded triple helices build up thecross-links (junction zones) in this network.When heating the gelatinsample the cross-links melt reversibly and absorbance decreases. Forthe wavenumber range below 1640 cm−1 a decrease in absorbance isobserved upon cooling and this is likely to correspond to the loss of β-sheet conformations of the gelatins, both intra- and intermolecularly.

To obtain Tg and Tm one can plot the intensity at a givenwavenumber or the shift of the amide I band maximum withtemperature. This is shown in Figs. 6 and 7, respectively. Only inFig. 7 can one observe a narrow transition curve, in agreement withprevious observations by Payne andVeis [30]. These plotswerefit witha sigmoid function to determine Tg and Tm of the samples (Table 2).

400 600 800 1000 1200

0

0.2

0.4

0.6

0.8

1

Ve(mL)

A /

Am

ax a

t 230

nm

85

110

125

130

145

Fig. 3. Fractionation of the gelatin coacervate from methanol coacervation on a semi-preparative Sephacryl S-400 HR column. Black ticks and fraction numbers show elutionpositions of selected tube fractions for FPLC.

6 8 10 12 14 16 18

0

0.2

0.4

0.6

0.8

1

1.2

Ve (mL)

A /

A m

ax a

t 214

nm

∼ α1

Fr∼ β∼ γMG

85 110 125 130 145

Fig. 4. FPLC analysis of selected tube fractions from the eluted coacervate (see Fig. 3).Abbrevations: Fr=fragments, MG=microgels.

ν (cm−1)

A

1600 1620 1640 1660 1680 17000

0.5

1

1.5

Fig. 5. Amide I band for a 5% (w/v) total gelatin in D2O during cooling (50 °C→5 °C).

321P. Chys et al. / Biochimica et Biophysica Acta 1814 (2011) 318–325

Interestingly, the melting temperature for α2 is significantly lowerthan Tm values for the other fractions and results also in a smallertemperature hysteresis. A difference in melting point Tm was alsodetermined in the experimental study of Harrington and Rao [31]. Intheir study the helix regeneration of α1 and α2 chains, obtained fromcollagen from three different species [rat skin, ichthyocol (glueprepared from the sounds of certain fishes) and cod skin], is examinedby means of optical rotation for dilute solutions. For ichthyocol,melting points for α1 and α2 are 34 °C and 32 °C, respectively. For therat skin sample, melting temperatures for α1 and α2 are 36 °C and35 °C, respectively. Apart from the difference in absolute values, it isclear that the difference in Tm for the two α fractions agrees very wellto the temperature difference determined in this work. The variationin temperature can be attributed to variation in amino acidcomposition (between the different species) as well as to the use ofD2O versus H2O [28].

To explain the difference between the fixed and variablewavenumber approach, we propose changes in the ν position torepresent the macroscopic phase transition (liquid→gel) whereasthe triple helix signal is probably not completely synchronous with

Table 1Peak assignment in the amide I band region of FTIR spectra of globular proteins, gelatins andanalysis. Data taken from [25–27] for globular proteins, from [28] for gelatin and from [29] fobond, PLP I=poly-L-proline type I helix (right-handed 103 helix), PLP II=poly-L-proline ty

ν (cm−1) Assignments

Globular [25–27] Gelatin [28]

1608 D2O-imide HB1616 IM AP β-sheet1621 β-sheet β-sheet/turns, D2O1632 β-sheet β-sheet, D2O-imide16351637 β-sheet

1641 310-helix1645 Unordered Random coil, D2O-g

16511653 β-helix1657 Triple helix, β-turn1663 Turns, bends1668 β-turns1671 β-turns1675 IM AP β sheet1680 β-turns1683 β-turns, IM AP β-sheet1689 Turns, bends

the macroscopic phase behavior. It is believed [32] that substantiallyless than 50% of the total possible helical content is needed to have aviscoelastic phase transition. Hence, a macroscopic transition canalready take placewhile the triple helix signal is only at the start of theS-shaped transition.

3.2.2. Difference spectraTo visualize the spectral changes more clearly, difference spectra

were constructed for all samples as a function of temperature.Difference spectra were made by subtraction of a reference spectrumfrom the baseline-corrected amide I bands. The spectrum at 50 °C and5 °C served as references for cooling and heating, respectively.

The difference spectra for α1 and α2 fraction, both upon cooling andheating, are similar (Fig. 8). Also no major differences were observedwith the spectra for coacervate and total gelatin (data not shown). Forall samples, on cooling a large increase occurs at 1657 cm−1 (triplehelix) whereas a substantial decrease occurs around 1623 cm−1 and1632 cm−1 (both β-sheet/turn [28] or PLP II-like helix [29]). The β-turnsignal around 1673 cm−1 also decreases. On heating, peaks withopposite sign are formed at the same wavenumbers. In addition, asubtle difference does seem to be present between the α1 samples (α1

poly-L-proline in D2O. The right column contains the results from the second derivativer poly-L-proline. Abbrevations: IM=intermolecular, AP=antiparallel, HB=hydrogen-pe II helix (left-handed 31 helix), PLP=poly-L-proline, PLHP=poly-L-hydroxyproline.

ν (cm−1)

Poly-L-proline [29] (this work)

16071615

-imide HB PLP II 1623HB PLP II-like (PLHP) 1632

PLP I

1640

lycine HB 16441649

PLP I–PLP II intermediate

s 16571664

1673

16831689

Table 2Gelation (Tg) and melting (Tm) temperature as determined by the wavenumberposition shifts during cooling and heating of 5% (w/v) gelatin samples. The hysteresis(ΔT) is the temperature difference between the obtained transition temperatures.

Sample Tg (°C) Tm (°C) ΔT (°C)

Total 24.3±1.2 32.8±0.6 8.5±1.3Coacervate 24.9±0.4 32.9±0.4 8.1±0.7α1 24.6±0.7 32.2±0.4 7.5±0.8α2 24.1±0.3 30.1±0.3 6.1±0.4

1600 1620 1640 1660 1680 1700

−0.3

−0.2

−0.1

0

0.1

0.2

ΔA (

a.u.

)

a

T (oC)

A a

t 162

1 cm

−1

0 10 20 30 40 501.2

1.25

1.3

1.35

1.4

1.45

Fig. 6. Change in absorbance at 1621 cm-1 for a 5% (w/v) total gelatin in D2O duringcooling (blue) and heating (red).

322 P. Chys et al. / Biochimica et Biophysica Acta 1814 (2011) 318–325

fraction and coacervate) and the α2-containing ones (α2 fraction andtotal gelatin) in the initial stage of cooling. The main changes describedabove are preceded by small and opposite ones which are slightly morepronounced in the α1-containing samples (wave-like spectra). Infraredabsorption decreases in the region 1640–1680 cm−1 whereas itincreases in the region 1600–1640 cm−1. The underlying phenomenaare not clear but the presence of opposite changes seems to suggestsome kind of conversion before main changes start to occur.Computation of the second derivative spectrum allowed identificationof the following peaks: 1607, 1615, 1623, 1632, 1640, 1644, 1649, 1657,1664, 1673, 1683 and 1689 cm−1. The calculated peak positions are ingood agreement with literature assignments (Table 1) [25–29]. Theminor peaks at 1640 and 1649 cm−1 are not assigned.

3.2.3. 2D COSThe one-dimensional analysis is further extended to obtain more

information on the sequence of structural events by 2D COS [22]. Here,an event is a change in intensity of the FTIR signal at a givenwavenumber ν, corresponding to a change of conformation of thegelatin. In the 2D COS triangles, each point relates the behavior of twotypes of conformational changes (e.g. β-turn versus triple helixchanges) during cooling and heating. The color intensity measures the

T (oC)

ν (c

m−

1 )

0 10 20 30 40 50 601650

1651

1652

1653

1654

1655

1656

Tg = 24 oC

Tm = 30 oC

Fig. 7. Fit (dashed line) of the evolution of the wavenumber position of the amide I bandmaximum. Sample is 5% (w/v) α2 fraction in D2O during cooling and heating(50 °C→5 °C → 50 °C).

degree of correlation between the two events. Application of the rulesof Noda [24] to the type of plot used here is as follows. A positive peakin the synchronous triangle means that the events at wavenumberX (X event) and wavenumber Y (Y event) change in the samedirection, a negative peak that events change oppositely. Examinationof the amide I band reveals now the absolute direction of the events.Corresponding cross-peaks in the asynchronous triangle are found byan isometrical reflection along the main diagonal, starting from thesynchronous cross-peaks or vice versa. If peaks have the same color,the X event in the synchronous triangle precedes the Y event. If thepeaks differ in sign, the X event of the synchronous peak lags the Yevent. If however, in the cross-examination of lower and highertriangles, the peaks of the asynchronous triangle are taken asreference the order of events reverts. For peaks with the same sign,the Y event of the asynchronous peak precedes the X event. For

ν (cm−1)

ν (cm−1)

ΔA (

a.u.

)

1600 1620 1640 1660 1680 1700

−0.3

−0.2

−0.1

0

0.1

0.2b

Fig. 8. Difference spectra for 5% (w/v) α1 fraction (a) and α2 fraction (b), calculatedfrom the original amide I band during a thermal cycle. Blue lines represent cooling, redlines heating.

Fig. 9. 2D COS plot of a 5% (w/v) α1 fraction in D2O during cooling (50 °C→5 °C). Lowertriangle represents the synchronous plot and the upper one is the asynchronous plot.See Section 2.2 for details.

Fig. 11. 2D COS plot of a 5% (w/v) α1 fraction in D2O during heating (5 °C→50 °C).

323P. Chys et al. / Biochimica et Biophysica Acta 1814 (2011) 318–325

differently colored peaks the reverse is true. As previously noticed(Section 2.2), for the upper triangle the Y event actually refers to the Xevent in the original asynchronous plot.

3.2.3.1. Cooling. The 2D COS plots during cooling for the α1 fraction(Fig. 9), α2 fraction (Fig. 10), coacervate and total gelatin are similar.The synchronous triangles are nearly identical, indicating the sameconformational changes for all samples. The asynchronous triangles doshow some different cross-peaks between α1 samples (α1 fraction andcoacervate) and the other ones. However, these peaks have no clearcounter-peaks in the synchronous triangle, indicating that it concernssmall amplitude events. Therefore, the basic sequence is the same for allsamples. The synchronous components have typical autopeaks around1623, 1656 and 1673 cm−1 (Fig. 10). Important cross-peaks are foundin the synchronous triangles at 1657/1623 cm−1 [− ,s/w], 1673/1657 cm−1 [− ,s] and 1673/1623 cm−1 [+,s/w], in agreement withthe difference spectra and confirming that the peaks around1623 cm−1 and 1673 cm−1 change oppositely to the main peak at1657 cm−1. The complementary asynchronous triangles offer insightinto the chronology of events. Cross-peaks at 1657(X)/1673(Y) cm−1

[− ,s], 1623/1673 cm−1 [+,s] and the lack of it at 1623/1657 cm−1

Fig. 10. 2D COS plot of a 5% (w/v) α2 fraction in D2O during cooling (50 °C→5 °C).

[− ,s] allow to determine the following basic sequence of eventsaccording to Ref. [24]:

1673↓→ 1657↑1623↓

Indeed, since the cross-peaks 1673/1657 cm−1 (synchronous) and1657/1673 cm−1 (asynchronous) are negative, 1673 cm−1 mustprecede 1657 cm−1. Also, since the synchronous cross-peak 1673/1623 cm−1 [+,s/w] has a positive counter-peak, 1673 cm−1 precedes1623 cm−1. Although cross-peak 1657/1623 cm−1 seems to have anasynchronous counter-peak, the exact peaks do not match. As a result,1657 cm−1 and 1623 cm−1 are in-phase events.

3.2.3.2. Heating. For the heating process, the 2D COS plots for the α1

fraction (Fig. 11), coacervate and total gelatin are almost identical.Their synchronous triangles show the same pattern as that observedfor all studied gelatin samples during cooling. Thus, despite theopposite intensity changes for cooling and heating of the amide Iband, the correlations between different wavenumber events arepreserved. The asynchronous triangles have cross-peaks 1657/1673 cm−1 [− ,s], 1623/1673 cm−1 [+,s/w], 1623/1657 cm−1 [+,s]and 1642/1657 cm−1 [+,s]. The latter is without counterpart in the

Fig. 12. 2D COS plot of a 5% (w/v) α2 fraction in D2O during heating (5 °C→50 °C).

324 P. Chys et al. / Biochimica et Biophysica Acta 1814 (2011) 318–325

synchronous triangle. Such cross-peaks have been observed previ-ously [33] and indicate that the two bands that contribute to thiscross-peak change out-of-phase with respect to each other. However,one cannot deduce any information about the sequence of eventsinvolving these two bands. It can be seen in the difference spectra thatthe changes at 1642 cm−1 do not correspond to any major peak, butrather to the edge of the triple helix band at 1657 cm−1 and the bandat 1623 cm−1. When the intensity of two overlapping bands changesin opposite direction such artificial cross-peaks are known to occur.Hence the 1642/1657 cm−1 cross-peak was not taken into account.Analysis yields the following sequence for heating of the α1 fraction:

1673↑→1623↑→1657↓

The 2D COS plot of the α2 fraction, however, is different from thatof the α1-containing fractions. While the synchronous triangle is stillthe same as for the other samples, the asynchronous triangle isdifferent (Fig. 12). From the cross-peaks with appropriate counter-parts in the synchronous triangle (1657/1673 cm−1 [− ,s], 1623/1673 cm−1 [+,w/s] and 1623/1657 cm−1 [− ,s]) the followingsequence is deduced for heating of α2:

1673↑→1657↓→1623↑

4. Discussion

The analysis of the amide I band region of gelatin revealed peakpositions in good agreement with other gelatin studies (Table 1).Nearly all of the peaks determined in the study by Prystupa andDonald [28] are confirmed. Payne and Veis [30] also studied analkali-processed gelatin albeit in H2O instead of D2O and assignpeak positions at 1633 cm−1, 1643 cm−1 and 1660 cm−1. Theseassignments are partially based on previous work of Lazarev andcoworkers [34] and are also in agreement with the positions here.The presence of a peak at 1673 cm−1 is consistent with Muyonga etal. [35] and the other three peak positions in this study correspondalso to positions determined here. Alternatively, the obtained peakpositions can be compared to typical assignments for globularproteins in D2O [25–27]. Good agreement is also found in suchcomparison (Table 1). The study of Dukor and Keiderling [29], amutarotation study of poly-L-proline and analogues by means ofFTIR and circular dichroism techniques, suggests alternativestructural assignments at 1623 cm-1 and 1632 cm-1. These authorsprimarily investigated poly-L-proline type I (PLP I) and type II (PLPII) conformations for a variety of solvents (also D2O). They alsostudied poly-L-hydroxyproline (PLHP) which is believed to assumea PLP II-like helix. Both poly-L-proline and poly-L-hydroxyprolineare biopolymers closely related to gelatin [4]. Most importantly,Dukor and Keiderling found peak positions at 1623 cm−1 for PLP IIhelices from PLP and 1632 cm−1 for PLP II-like helices from PLHP inthe FTIR measurements. The assignments at 1623 and 1632 cm−1

remain to be elucidated in future work.Our 2D COS analyses show the same main events on cooling for all

examined samples. They indicate the disappearance of the band at1673 cm−1 (β-sheets/turns) to trigger triple helix formation. The 2DCOS analyses thus support the β-turn mechanisms proposed byBusnel et al. [36] and Prystupa and Donald [28]. This corresponds tothe following gelation model. At 50 °C transient β-turns (1673 cm−1)are present in gelatin chains. Upon cooling transient β-turns alterconformation and allow collagen-folds to be formed. In a collagen-fold, two separate and helical gelatin segments are aligned in anantiparallel manner [4]. It principally results from an intramolecularevent and constitutes a metastable intermediate. These collagen-foldsare now predecessors and nucleation sites for triple helix formation.By rapid bimolecular association with gelatin chain segments in

solution, regenerated triple helix segments develop which form thecross-links in the gel network. The rate limiting gelation model fromBusnel et al. [36] in which intramolecular nucleation is followed bybimolecular nucleation at sufficiently high concentration is thussupported by our data. The event changes at 1673 cm−1 pointtowards the involvement of β-turns and intramolecular phenomenawhereas the coincident absorption decrease at 1623 cm−1 (β-sheet/PLP II helix) and increase at 1657 cm−1 (triple helices) support therapid bimolecular step. The model of Busnel et al. [36] goes furtherback to the Flory and Weaver [37] model in which the two-stepkinetic mechanism was first introduced. This model already pointedtowards the involvement of turn structures in the first step butassumed the second step to be trimolecular.

On heating, a difference is revealed by the 2D COS analysesbetween the α1 fraction, coacervate and total gelatin on the one hand,and the α2 fraction on the other hand. Since coacervate and totalgelatin mainly consist of α1 chains it is plausible that these samplesbasically behave like the α1 fraction. In all cases the heating processstarts with an increase of the band at 1673 cm−1, likely pointing out adestabilisation of the collagen-fold as well as indicating paralleldevelopment of β-turns in segments which have remained freeduring gelation. In [38] it is shown that collagen-folds in triple helicesdestabilise upon reaching temperatures close to the melting temper-ature and that this occurs before strand separation. This is also astrong argument to assume that collagen-folds are the intermediateinstead of single chains as is the case in the model of Prystupa andDonald [28]. The fact that changes at 1673 cm−1 precede otherspectral changes both upon heating and cooling can also be explainedby the fact that triple helices upon melting become more thermallystable.

Specifically for α2, the loss of triple helices precedes the increase at1623 cm−1. This could indicate that destabilisation of the collagen-fold immediately leads to destabilisation of the triple helix. The triplehelix separates and frees gelatin chains. It seems that in case of α2 thecollagen-folds are stabilised in a strong cooperative way by a third α2

chain.However, for α1 (like for coacervate and total gelatin) changes at

1623 cm−1 precede the decrease at 1657 cm−1 (triple helix).Appearance of β-sheets or PLP II-like gelatin structures would occurbefore the triple helix disappears. Since pure α1 samples are present inthis group, the effect does not result from the presence of α2 chainswith lower thermal stability. It seems most plausible that destabilisa-tion of the collagen folds of α1 does not lead to completedestabilisation of the triple helix and that this allows for localgelation/melting cycles until this is no longer possible at the truemelting point.

5. Conclusions

In this FTIR study of an alkali-pretreated gelatin we investigatedisolated α fractions, obtained by methanol coacervation and semi-preparative gel chromatography. Samples were subjected to thethermal cycle 50 °C→5 °C → 50 °C. The estimated gelation tempera-tures show no differences for the different samples (24.5 °C).However, the melting temperatures do differ and the α2 fraction hasa melting temperature of 2 °C lower (30 °C) than the other samples(total, coacervate and α1 fraction). 2D COS and difference spectrashow that on cooling all samples have very similar structuraldynamics. However, on heating the α2 fraction deviates from theother samples by an earlier loss of triple helical structures in theconformational cascade which correlates with the determined lowerthermostability of a gel consisting uniquely of α2 chains (lower Tm).2D COS also shows that for all examined samples alterations in β-turnconformation start the sequence of conformational events both uponcooling and heating.

325P. Chys et al. / Biochimica et Biophysica Acta 1814 (2011) 318–325

Acknowledgments

We are grateful to Niek Hias for his assistance with theexperiments. We are greatly indebted to PB Gelatins (TessenderloChemie, Vilvoorde, Belgium) for the delivery of the gelatin sample. F.M. is a postdoctoral fellow of the Research Foundation Flanders (FWO-Vlaanderen).

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