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

Biochimica et Biophysica Acta 1814 (2011) 318–325

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a p a p

FTIR 2D correlation spectroscopy of α1 and α2 fractions of an alkali-pretreated gelatin Pieter Chys a, Constant Gielens a, Filip Meersman b,⁎ a b

Katholieke Universiteit Leuven, Department of Chemistry, Division Biochemistry, Molecular and Structural Biology, Celestijnenlaan 200 G, 3001 Leuven, Belgium Division Molecular and Nanomaterials, Celestijnenlaan 200 F, 3001 Leuven, Belgium

a r t i c l e

i n f o

Article history: Received 15 February 2010 Received in revised form 24 September 2010 Accepted 4 October 2010 Available online 13 October 2010 Keywords: Gelatin α fraction FTIR spectroscopy Alcohol coacervation Fractionation Sol–gel transition 2D COS

a b s t r a c t An alkali-pretreated gelatin (pI ~ 4.9) was fractionated by means of alcohol coacervation and semi-preparative gel chromatography. The thermal responses of the isolated α fractions, the coacervate and the total gelatin were investigated by 2D-correlation FTIR spectroscopy in the amide I band region (1600–1700 cm− 1). The gelation temperature was the same for all examined samples (24.5 °C) while the melting temperature of the α2 fraction was lower (30 °C) than that of the other samples (32.5 °C). The 2D COS plots indicate that on cooling (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 the triple helix signal in the event sequence. The lower melting temperature (less thermostable gelatin gel) of the α2 fraction thus results from a different conformational cascade of the α2 chains upon melting. In all samples the initial conformational changes take place in the β-turns, providing further evidence for the models proposed previously. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Gelatins are denatured collagen molecules with a wide range of applications [1,2]. The collagen molecule is one of the most important structural proteins in the animal kingdom [3]. A variety of gelatins exists originating from different collagen types, manufacturing protocols, Mr distributions and so on [4,5]. From the economic point of view, gelatins account for a substantial amount of the biomaterial traded in the food industry [6]. The physicochemical nature of gelatins is unique [4,5]. Gelatins behave completely different in solution as compared to globular proteins and are random-coil like and extended instead of native and compact. A high degree of monodispersity is difficult to attain for manufactured gelatins and most gelatin samples cover quite a broad Mr range [4,5,7], which is both a strength and drawback. Depending on 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 highly dependent on the pH of the solution [8–13]. The above features result in the ability of gelatin solutions to form physical gels when the gelatin concentration is high enough [4,5]. Such gelatin gels exhibit a viscoelastic sol–gel transition and are thermoreversible in nature. However, gelation (Tg) and melting (Tm) temperatures for the same ⁎ Corresponding author. Tel.: + 32 16327355; fax: + 32 16327990. E-mail addresses: [email protected] (P. Chys), [email protected] (C. Gielens), [email protected] (F. Meersman). 1570-9639/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2010.10.003

gelatin gel mostly differ and result in temperature hysteresis of experimental cooling (gelation) and heating (melting) curves, indicating a different stability upon heating and cooling of the gelatin gel [4]. The looseness and randomness of the gelatin architecture in solution is in sharp contrast to the native structure of collagen: a stiff triple helix (tropocollagen) of three α chains [5,14]. The constituting α chains (Mr ~ 95 000) remain the building blocks for gelatins but all geometric constraints on conformation, such as present in tropocollagen, are lost in solution. In addition, the α chains are not always the same in a single tropocollagen molecule of a given biological species [3,15]. At present, approximately 30 types of tropocollagen are known of which some consist of a single and unique α chain, whereas others do not [16]. For example, in collagen type I, the main source for gelatins in industry, two α1 chains and one α2 chain, build up the tropocollagen macromolecule. The purpose of the study here is to examine in detail the thermal behavior of α1 and α2 fractions and to investigate whether or not these α fractions behave differently. To this end α1 and α2 chains from a total alkali-pretreated gelatin were fractionated by means of methanol coacervation and semi-preparative gel chromatography. The fractions were subsequently characterized by one- and twodimensional Fourier transform infrared (FTIR) correlation spectroscopy (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 kindly provided by PB Gelatins, Tessenderlo Chemie (Vilvoorde, Belgium).

P. 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 five fractions [17]. The principal fraction is the α fraction which has two subfractions [5], α1 and α2, which originate from the α chains constituting collagen type I. The β and γ fractions are double and triple covalently cross-linked α chains, respectively. Microgels (Mr N N 106) 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 of simple alcohol coacervation and semi-preparative gel chromatography. 2.1.1. Coacervation The coacervation protocol is modified from Veis [4]. Gelatin grains were added to an aqueous 0.8 M NaCl solution at 2% (w/v) and kept for 1.5 h at room temperature (22 °C) to allow for swelling. This mixture 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 warm gelatin solution. After homogenization the mixture was allowed to cool and kept overnight at room temperature (15–16 h). The supernatant, consisting of the α2 fraction, was decanted and lyophilized at –30 °C to remove the methanol. The dry product was redissolved in ultrapure water, dialyzed against ultrapure water and lyophilized again. The coacervate was appropriately diluted to obtain a solution and heated after which it was also lyophilized. 2.1.2. Gel chromatography To 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 HR column (86 cm × 4.0 cm) and eluted. The column was thermostatted at 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. Flow was 0.8 mL/min and separation range of the column is 2 × 10 4 −8 × 10 6 Da for globular proteins (1 × 10 4 −2 × 106 Da for linear dextrans). After location of the α1 chains in the eluate, selected tube fractions were collected and lyophilized. Gelatin samples were identified by means of Fast Protein Liquid Chromatography (FPLC) on a Superose 6 HR 10/35 column (Amersham Biosciences). The column has a separation range for globular proteins from 5 × 103 to 5 × 106 Da (exclusion limit ~ 4 × 107 Da). Flow was 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 and 0.15 M NaCl. Working pressure was 0.8–0.9 MPa. FPLC was also used to determine the composition of the total gelatin sample for reference. This sample was made as described above but without the coacervation step. For all samples, after spectrophotometric determination of gelatin concentration (a230 = 2.0 L/g·cm [17]), an appropriate dilution [0.3% (w/v)] was made and the samples were heated for 1 h at 50 °C. A volume of 300–600 μL of the warm solution was injected. 2.1.3. Electrophoresis Sodium dodecyl sulphate electrophoresis (SDS-PAGE) allows the discrimination between gelatin α1 and α2 fractions [19]. The electrophoresis was performed on an LKB 2050 Midget apparatus using a current of 25 mA per gel. Stacking gel was 4% (w/v) polyacrylamide and running gel 7.5% (w/v) polyacrylamide. Electrophoresis 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 solution volume was larger than 10 μL, the sample was first dried with a speedvac concentrator (SC 110 AR, Savant). To the sample, 20 μL sample buffer, containing 2% (w/v) SDS and 1% (v/v) 2-mercaptoethanol, was

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added. This mixture was heated at 100 °C for 3–4 min. After the electrophoresis, the gel was carefully removed from the glass plates and placed for 30 min in a coloring solution of Coomassie Brilliant Blue 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 were cooled down to 5 °C and upon reaching 5 °C, the samples were immediately reheated to 50 °C. Cooling and heating rate was 0.2 °C/ min. One-dimensional spectra [20,21] were obtained with a Bruker IFS66 Spectrometer equipped with a liquid nitrogen cooled mercurycadmium-telluride (MCT) detector. The sample compartment was continuously purged with dry air. At each temperature, 256 interferograms (resolution 2 cm− 1) were averaged for each spectrum. The temperature cell is made up by two CaF2 windows and a 50 μm teflon spacer, and is controlled by an Automatic Temperature Controller of Graseby Specac. Two-dimensional correlation spectroscopy (2D COS) of the onedimensional FTIR spectra was implemented in MATLAB [22]. A single window over the entire temperature range of the experiment (5 °C−50 °C) was used. From the complete series of FTIR spectra as a function of temperature, the raw data of the amide I band were taken, smoothed twice by spline interpolation and a linear background (1590–1700 cm− 1) subtracted. To check the validity of the baseline correction a wider wavenumber range (1400−1900 cm− 1) was tested in 2D COS calculations but this made no difference and therefore the narrow range was used. Next, the dynamic component of the FTIR spectra was calculated by subtraction of the static component. Since subtraction of an average static spectrum can amplify noise and introduce false peaks when the dynamic component has relatively low amplitude versus the static component [23], we used the steady-state spectrum as reference. For cooling this was the 50 °C spectrum, for heating the 5 °C spectrum. Since 2D COS plots with no scaling yielded good results, we kept the computational procedure as simple as possible and avoided scaling techniques. No scaling was thus done after computation of the dynamic component. The discrete Fourier transform and its conjugate transform for a given structural event pair (e.g. change at ν1 versus change at ν2) could now be calculated and separately summed. A complex number for each possible pair of wavenumbers (ν1,ν2) is obtained: Φ + Ψi. The real component Φ of this number represents the synchronous component of the correlation, the imaginary part Ψ the asynchronous component. From the real and imaginary part of Φ + Ψi, synchronous and asynchronous plots are constructed and interpreted according to the rules of Noda [24]. We introduce a graphical representation which shows the information of both separate component plots in one, with the synchronous information in the lower triangle and the asynchronous in the upper triangle. Construction of such a graph is as follows. The lower triangle of the synchronous plot is first plotted at its normal place on an empty 2D COS grid. Hereafter, the lower triangle of the asynchronous plot is orthogonally reflected along its main diagonal and plotted into the upper triangular area. Thus, the Y axis of the new 2D COS plot corresponds to the original X axis of the asynchronous plot in case of the asynchronous (upper) triangle. Since both the lower triangles of the component plots are used, the rules of Noda [24] remain valid. This type of graph has the advantage that the data redundancy in both separate plots is eliminated. Since the asynchronous component was much smaller in general, we scaled the asynchronous component data range towards the range of the synchronous component for uniformity of the color scale. For both the synchronous and asynchronous triangles, cross-peaks are referred

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to by using X/Y cm− 1 [−,s], with X and Y the associated wavenumber positions at the horizontal and vertical axes of the 2D COS plot. Thus, 1657/1623 cm− 1 refers to the cross-peak in the synchronous triangle whereas 1623/1657 cm− 1 refers to its counterpeak in the asynchronous triangle. Additional information in between brackets is given for the 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 (solid blue) as well as a Gaussian polyfit for 6 fractions. The fragments were divided in two fractions thereby yielding a sixth fraction. The α fraction (red dashed) at 11.5 mL is the most important fraction, consisting of both the α1 and α2 fractions, which have an identical molecular mass. The SDS-PAGE from total gelatin, supernatant and coacervate after the methanol coacervation is shown in Fig. 2. In electrophoresis lane 1 (total gelatin) two bands are observed near the α position corresponding to the two α fractions. Electrophoresis results in Aoyagi et al. [19] show the upper band to be α1 and the lower α2. The SDS-PAGE reveals that α1 enters mainly the coacervate whereas α2 remains mostly in the supernatant. This electrophoresis demonstrates that the two α fractions are largely separated by the methanol coacervation. Thus, the supernatant of an appropriate coacervation yields 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 from the methanol coacervation (Fig. 3) shows a broad band and no clear distinct peaks for the different fractions of the coacervate. However, samples taken over the complete elution range (black and numbered ticks in Fig. 3) and subjected to FPLC show that the selected fractions have different compositions (Fig. 4). Tube fraction 130 can be identified as corresponding to an enriched α fraction (see Fig. 1) and, since the coacervate is mainly devoid of α2 this fraction constitutes enriched α1 fraction. 3.2. FTIR spectroscopy FTIR spectroscopy was used to determine the gelation (Tg) and melting (Tm) temperatures, and mechanistic insight into the sol–gel transition was obtained by a 2D COS analysis. The changes in

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

secondary structure were monitored through the conformationally sensitive amide I band that results from the carbonyl groups in proteins and is strongly influenced by the local surrounding and dipole coupling to other carbonyl groups [20,21]. Table 1 shows, next to the empirical secondary structure–wavenumber correlations found for globular proteins [25–27], also assignments proposed for gelatin [28] and poly-L-proline [29]. 3.2.1. Gelation and melting point Fig. 5 shows the thermal response of the amide I band for a total gelatin sample during cooling. Around 1657 cm− 1 the peak increases upon cooling, indicating the formation of triple helix structure in agreement with previous observations [28,30]. Upon sufficient cooling and subsequent gelation an infinite gelatin network forms and it is generally assumed that partially refolded triple helices build up the cross-links (junction zones) in this network. When heating the gelatin sample the cross-links melt reversibly and absorbance decreases. For the wavenumber range below 1640 cm− 1 a decrease in absorbance is observed upon cooling and this is likely to correspond to the loss of βsheet conformations of the gelatins, both intra- and intermolecularly. To obtain Tg and Tm one can plot the intensity at a given wavenumber or the shift of the amide I band maximum with temperature. This is shown in Figs. 6 and 7, respectively. Only in Fig. 7 can one observe a narrow transition curve, in agreement with previous observations by Payne and Veis [30]. These plots were fit with a sigmoid function to determine Tg and Tm of the samples (Table 2).

0.6

1

total A / Amax at 230 nm

A at 214 nm

0.5

0.4

α

0.3

0.2 micro− gels

γ

β

0.8

0.6

0.4

0.2

85

0.1

fragments 1

125 110

fragments 2

0

400 6

8

10

12

14

145

0

16

18

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

600

130 800

1000

1200

Ve(mL) Fig. 3. Fractionation of the gelatin coacervate from methanol coacervation on a semipreparative Sephacryl S-400 HR column. Black ticks and fraction numbers show elution positions of selected tube fractions for FPLC.

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

1

85

110

Fr ∼β ∼γ MG

125 130 145

1 0.8

1

0.6

A

A / A max at 214 nm

1.5

∼α

1.2

321

0.4 0.5

0.2 0 6

8

10

12

14

16

0 1600

18

1620

1640

1660

1680

1700

ν (cm−1)

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

Interestingly, the melting temperature for α2 is significantly lower than Tm values for the other fractions and results also in a smaller temperature hysteresis. A difference in melting point Tm was also determined in the experimental study of Harrington and Rao [31]. In their study the helix regeneration of α1 and α2 chains, obtained from collagen from three different species [rat skin, ichthyocol (glue prepared from the sounds of certain fishes) and cod skin], is examined by means of optical rotation for dilute solutions. For ichthyocol, melting points for α1 and α2 are 34 °C and 32 °C, respectively. For the rat skin sample, melting temperatures for α1 and α2 are 36 °C and 35 °C, respectively. Apart from the difference in absolute values, it is clear that the difference in Tm for the two α fractions agrees very well to the temperature difference determined in this work. The variation in temperature can be attributed to variation in amino acid composition (between the different species) as well as to the use of D2O versus H2O [28]. To explain the difference between the fixed and variable wavenumber approach, we propose changes in the ν position to represent the macroscopic phase transition (liquid → gel) whereas the triple helix signal is probably not completely synchronous with

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

the macroscopic phase behavior. It is believed [32] that substantially less than 50% of the total possible helical content is needed to have a viscoelastic phase transition. Hence, a macroscopic transition can already take place while the triple helix signal is only at the start of the S-shaped transition. 3.2.2. Difference spectra To 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 spectrum from the baseline-corrected amide I bands. The spectrum at 50 °C and 5 °C served as references for cooling and heating, respectively. The difference spectra for α1 and α2 fraction, both upon cooling and heating, are similar (Fig. 8). Also no major differences were observed with the spectra for coacervate and total gelatin (data not shown). For all samples, on cooling a large increase occurs at 1657 cm− 1 (triple helix) whereas a substantial decrease occurs around 1623 cm− 1 and 1632 cm− 1 (both β-sheet/turn [28] or PLP II-like helix [29]). The β-turn signal around 1673 cm− 1 also decreases. On heating, peaks with opposite sign are formed at the same wavenumbers. In addition, a subtle difference does seem to be present between the α1 samples (α1

Table 1 Peak assignment in the amide I band region of FTIR spectra of globular proteins, gelatins and poly-L-proline in D2O. The right column contains the results from the second derivative analysis. Data taken from [25–27] for globular proteins, from [28] for gelatin and from [29] for poly-L-proline. Abbrevations: IM = intermolecular, AP = antiparallel, HB = hydrogenbond, PLP I = poly-L-proline type I helix (right-handed 103 helix), PLP II = poly-L-proline type II helix (left-handed 31 helix), PLP = poly-L-proline, PLHP = poly-L-hydroxyproline. ν (cm− 1)

Globular [25–27] 1608 1616 1621 1632 1635 1637

ν (cm− 1)

Assignments Gelatin [28]

Poly-L-proline [29]

D2O-imide HB IM AP β-sheet β-sheet β-sheet

β-sheet/turns, D2O-imide HB β-sheet, D2O-imide HB

PLP II PLP II-like (PLHP) PLP I

(this work) 1607 1615 1623 1632

β-sheet 1640

1641 1645 1651 1653 1657 1663 1668 1671 1675 1680 1683 1689

310-helix Unordered

Random coil, D2O-glycine HB

1644 1649 PLP I–PLP II intermediate

β-helix Triple helix, β-turns Turns, bends

1657 1664

β-turns β-turns IM AP β sheet

1673 β-turns

β-turns, IM AP β-sheet Turns, bends

1683 1689

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1.45

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

1.35

Sample

Tg (°C)

Tm (°C)

ΔT (°C)

Total Coacervate α1 α2

24.3 ± 1.2 24.9 ± 0.4 24.6 ± 0.7 24.1 ± 0.3

32.8 ± 0.6 32.9 ± 0.4 32.2 ± 0.4 30.1 ± 0.3

8.5 ± 1.3 8.1 ± 0.7 7.5 ± 0.8 6.1 ± 0.4

1.3

1.25

1.2

0

10

20

T Fig. 6. Change in absorbance at 1621 cm cooling (blue) and heating (red).

-1

30 o ( C)

40

50

for a 5% (w/v) total gelatin in D2O during

fraction and coacervate) and the α2-containing ones (α2 fraction and total gelatin) in the initial stage of cooling. The main changes described above are preceded by small and opposite ones which are slightly more pronounced in the α1-containing samples (wave-like spectra). Infrared absorption decreases in the region 1640–1680 cm− 1 whereas it increases in the region 1600–1640 cm− 1. The underlying phenomena are not clear but the presence of opposite changes seems to suggest some kind of conversion before main changes start to occur. Computation of the second derivative spectrum allowed identification of the following peaks: 1607, 1615, 1623, 1632, 1640, 1644, 1649, 1657, 1664, 1673, 1683 and 1689 cm− 1. The calculated peak positions are in good agreement with literature assignments (Table 1) [25–29]. The minor peaks at 1640 and 1649 cm− 1 are not assigned. 3.2.3. 2D COS The 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 given wavenumber ν, corresponding to a change of conformation of the gelatin. In the 2D COS triangles, each point relates the behavior of two types of conformational changes (e.g. β-turn versus triple helix changes) during cooling and heating. The color intensity measures the

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

a 0.2

0.1

ΔA (a.u.)

A at 1621 cm−1

1.4

0

−0.1

−0.2

−0.3 1600

1620

1640

1680

1700

ν

1660 −1 (cm )

1640

1660

1680

1700

b

1656

0.2

Tg = 24 oC Tm = 30 oC

1655

0.1

ΔA (a.u.)

ν (cm−1)

1654

1653

0

−0.1

1652 −0.2 1651 −0.3 1650

0

10

20

30

40

50

60

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

1600

1620

ν (cm−1) Fig. 8. Difference spectra for 5% (w/v) α1 fraction (a) and α2 fraction (b), calculated from the original amide I band during a thermal cycle. Blue lines represent cooling, red lines heating.

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

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

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

323

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

[−,s] allow to determine the following basic sequence of events according to Ref. [24]: 1673↓→ 1657↑ 1623↓

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 same conformational changes for all samples. The asynchronous triangles do show some different cross-peaks between α1 samples (α1 fraction and coacervate) and the other ones. However, these peaks have no clear counter-peaks in the synchronous triangle, indicating that it concerns small amplitude events. Therefore, the basic sequence is the same for all samples. The synchronous components have typical autopeaks around 1623, 1656 and 1673 cm− 1 (Fig. 10). Important cross-peaks are found in the synchronous triangles at 1657/1623 cm− 1 [−,s/w], 1673/ 1657 cm− 1 [−,s] and 1673/1623 cm− 1 [+,s/w], in agreement with the difference spectra and confirming that the peaks around 1623 cm− 1 and 1673 cm− 1 change oppositely to the main peak at 1657 cm− 1. The complementary asynchronous triangles offer insight into 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

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

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

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

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 observed for all studied gelatin samples during cooling. Thus, despite the opposite intensity changes for cooling and heating of the amide I band, the correlations between different wavenumber events are preserved. 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

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synchronous triangle. Such cross-peaks have been observed previously [33] and indicate that the two bands that contribute to this cross-peak change out-of-phase with respect to each other. However, one cannot deduce any information about the sequence of events involving these two bands. It can be seen in the difference spectra that the changes at 1642 cm− 1 do not correspond to any major peak, but rather to the edge of the triple helix band at 1657 cm− 1 and the band at 1623 cm− 1. When the intensity of two overlapping bands changes in 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 that of the α1-containing fractions. While the synchronous triangle is still the same as for the other samples, the asynchronous triangle is different (Fig. 12). From the cross-peaks with appropriate counterparts in the synchronous triangle (1657/1673 cm− 1 [− ,s], 1623/ 1673 cm− 1 [+,w/s] and 1623/1657 cm− 1 [− ,s]) the following sequence is deduced for heating of α2: 1673↑→1657↓→1623↑

4. Discussion The analysis of the amide I band region of gelatin revealed peak positions in good agreement with other gelatin studies (Table 1). Nearly all of the peaks determined in the study by Prystupa and Donald [28] are confirmed. Payne and Veis [30] also studied an alkali-processed gelatin albeit in H2O instead of D2O and assign peak positions at 1633 cm− 1, 1643 cm− 1 and 1660 cm− 1. These assignments are partially based on previous work of Lazarev and coworkers [34] and are also in agreement with the positions here. The presence of a peak at 1673 cm− 1 is consistent with Muyonga et al. [35] and the other three peak positions in this study correspond also to positions determined here. Alternatively, the obtained peak positions can be compared to typical assignments for globular proteins in D2O [25–27]. Good agreement is also found in such comparison (Table 1). The study of Dukor and Keiderling [29], a mutarotation study of poly-L-proline and analogues by means of FTIR and circular dichroism techniques, suggests alternative structural assignments at 1623 cm-1 and 1632 cm-1. These authors primarily investigated poly-L-proline type I (PLP I) and type II (PLP II) conformations for a variety of solvents (also D2O). They also studied poly-L-hydroxyproline (PLHP) which is believed to assume a PLP II-like helix. Both poly-L-proline and poly-L-hydroxyproline are biopolymers closely related to gelatin [4]. Most importantly, Dukor and Keiderling found peak positions at 1623 cm− 1 for PLP II helices from PLP and 1632 cm− 1 for PLP II-like helices from PLHP in the 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 at 1673 cm− 1 (β-sheets/turns) to trigger triple helix formation. The 2D COS analyses thus support the β-turn mechanisms proposed by Busnel et al. [36] and Prystupa and Donald [28]. This corresponds to the following gelation model. At 50 °C transient β-turns (1673 cm− 1) are present in gelatin chains. Upon cooling transient β-turns alter conformation and allow collagen-folds to be formed. In a collagenfold, two separate and helical gelatin segments are aligned in an antiparallel manner [4]. It principally results from an intramolecular event and constitutes a metastable intermediate. These collagen-folds are 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 the cross-links in the gel network. The rate limiting gelation model from Busnel et al. [36] in which intramolecular nucleation is followed by bimolecular nucleation at sufficiently high concentration is thus supported by our data. The event changes at 1673 cm− 1 point towards the involvement of β-turns and intramolecular phenomena whereas the coincident absorption decrease at 1623 cm− 1 (β-sheet/ PLP II helix) and increase at 1657 cm− 1 (triple helices) support the rapid bimolecular step. The model of Busnel et al. [36] goes further back to the Flory and Weaver [37] model in which the two-step kinetic mechanism was first introduced. This model already pointed towards the involvement of turn structures in the first step but assumed the second step to be trimolecular. On heating, a difference is revealed by the 2D COS analyses between the α1 fraction, coacervate and total gelatin on the one hand, and the α2 fraction on the other hand. Since coacervate and total gelatin mainly consist of α1 chains it is plausible that these samples basically behave like the α1 fraction. In all cases the heating process starts with an increase of the band at 1673 cm− 1, likely pointing out a destabilisation of the collagen-fold as well as indicating parallel development of β-turns in segments which have remained free during gelation. In [38] it is shown that collagen-folds in triple helices destabilise upon reaching temperatures close to the melting temperature and that this occurs before strand separation. This is also a strong argument to assume that collagen-folds are the intermediate instead of single chains as is the case in the model of Prystupa and Donald [28]. The fact that changes at 1673 cm− 1 precede other spectral changes both upon heating and cooling can also be explained by the fact that triple helices upon melting become more thermally stable. Specifically for α2, the loss of triple helices precedes the increase at 1623 cm− 1. This could indicate that destabilisation of the collagenfold immediately leads to destabilisation of the triple helix. The triple helix separates and frees gelatin chains. It seems that in case of α2 the collagen-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 occur before the triple helix disappears. Since pure α1 samples are present in this group, the effect does not result from the presence of α2 chains with lower thermal stability. It seems most plausible that destabilisation of the collagen folds of α 1 does not lead to complete destabilisation of the triple helix and that this allows for local gelation/melting cycles until this is no longer possible at the true melting point.

5. Conclusions In this FTIR study of an alkali-pretreated gelatin we investigated isolated α fractions, obtained by methanol coacervation and semipreparative gel chromatography. Samples were subjected to the thermal cycle 50 °C → 5 °C → 50 °C. The estimated gelation temperatures show no differences for the different samples (24.5 °C). However, the melting temperatures do differ and the α2 fraction has a melting temperature of 2 °C lower (30 °C) than the other samples (total, coacervate and α1 fraction). 2D COS and difference spectra show that on cooling all samples have very similar structural dynamics. However, on heating the α2 fraction deviates from the other samples by an earlier loss of triple helical structures in the conformational cascade which correlates with the determined lower thermostability of a gel consisting uniquely of α2 chains (lower Tm). 2D COS also shows that for all examined samples alterations in β-turn conformation start the sequence of conformational events both upon cooling and heating.

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Acknowledgments We are grateful to Niek Hias for his assistance with the experiments. We are greatly indebted to PB Gelatins (Tessenderlo Chemie, Vilvoorde, Belgium) for the delivery of the gelatin sample. F. M. is a postdoctoral fellow of the Research Foundation Flanders (FWOVlaanderen).

References [1] H. Boedtker, P. Doty, A study of gelatin molecules, aggregates and gels, J. Phys. Chem. 58 (1954) 968–983. [2] B. Mohanty, A. Gupta, H.B. Bohidar, S. Bandyopadhyay, Effect of gelatin molecular charge heterogeneity on formation of intermolecular complexes and coacervation transition, J. Polym. Sci. B Polym. Phys. 45 (2007) 1511–1520. [3] B.J. Runnegar, Collagen gene construction and evolution, Mol. Evol. 22 (1985) 141–149. [4] A. Veis, The Macromolecular Chemistry of Gelatin, Academic Press, New York, 1964. [5] P.J. Rose, Inedible gelatin and glue, in: A.M. Pearson, T.R. Dutson (Eds.), Inedible Meat By-Products, Advances in Meat Research, Elsevier Applied Science, New York, 1992, pp. 217–263. [6] R.L. Whistler, J.N. Bemiller, Carbohydrate Chemistry for Food Scientists, Eagon Press, 1997. [7] M. Meyer, B. Morgenstern, Characterization of gelatin and acid soluble collagen by size exclusion chromatography coupled with multi angle light scattering (SECMALS), Biomacromolecules 4 (2003) 1727–1732. [8] A.V. Dobrynin, R.H. Colby, M.J. Rubinstein, Polyampholytes J. Polym. Sci. B Polym. Phys. 42 (2004) 3513–3538. [9] A.V. Dobrynin, M. Rubinstein, Theory of polyelectrolytes in solutions and at surfaces, Progr. Polym. Sci. 30 (2005) 1049–1118. [10] M.C. Barbosa, Y. Levin, Phase transition of a neutral polyampholyte, Phys. A 231 (1996) 467–483. [11] E.Y. Kramarenko, A.R. Khokhlov, N. Yoshikawa, A three-state model for counterions in a dilute solution of weakly charged polyelectrolytes, Macromol. Theory Simul. 9 (2000) 249–256. [12] A. Kudlay, M.O. de la Cruz, Precipitation of oppositely charged polyelectrolytes in salt solutions, J. Chem. Phys. 120 (2004) 404–412. [13] D.W. Cheong, A.Z. Panagiotopoulos, Phase behaviour of polampholyte chains from grand canonical Monte Carlo simulations, Mol. Phys. 103 (2005) 3031–3044. [14] R.C. Cantor, P.R. Schimmel, Biophysical Chemistry, The Behaviour of Biological Macromolecules, Volume 3, W.H. Freeman and Company, San Francisco, 1980. [15] E.J. Miller, S. Gay, Dimensions of protein random coils, Meth. Enzymol. 144 (1987) 3–41. [16] B. Brodsky, G. Thiagarajan, B. Madhan, K. Kar, Triple-helical peptides: an approach to collagen conformation, stability, and self-association, Biopolymers 89 (2008) 345–353.

325

[17] E. van den Bosch, C. Gielens, Gel chromatographic fractionation and partial physicochemical characterization of gelatins, Chromatographia 58 (2003) 507–511. [18] E. van den Bosch, C. Gielens, Gelatin degradation at elevated temperature, Int. J. Biol. Macromol. 32 (2003) 129–138. [19] S. Aoyagi, N. Ohkubo, Y.J. Otsubo, Alcohol precipitation for the separation of α1 and α2 components of gelatin, Imaging Sci. 45 (1997) 178–182. [20] J.L.R. Arrondo, A. Muga, J. Castresana, F.M. Goñi, Qualitative studies of the structure of proteins in solution by Fourier-Transform infrared spectroscopy, Prog. Biophys. Molec. Biol. 59 (1993) 23–56. [21] P.I. Haris, D. Chapman, The conformational analysis of peptides using Fourier Transform IR spectroscopy, Biopolymers 37 (1995) 251–263. [22] P.B. Harrington, A. Urbas, P.J. Tandler, Two-dimensional correlation analysis, Chemom. Intell. Lab. Syst. 50 (2000) 149–174. [23] P.J. Tandler, P.de.B. Harrington, H. Richardson, Effects of static spectrum removal and noise on 2D-correlation spectra of kinetic data, Anal. Chim. Acta 368 (1998) 45–57. [24] I. Noda, Two-dimensional infrared (2D IR) spectroscopy: theory and applications, Appl. Spectrosc. 44 (1990) 550–561. [25] D.M. Byler, H. Susi, Examination of the secondary structure of proteins by deconvolved FTIR spectra, Biopolymers 25 (1986) 469–487. [26] M. Jackson, H.H. Mantsch, The use and misuse of FTIR spectroscopy in the determination of protein structure, Crit. Rev. Biochem. Mol. Biol. 30 (1995) 95–120. [27] F. Meersman, L. Smeller, K. Heremans, Comparative Fourier transform infrared spectroscopy study of cold-, pressure-, and heat-induced unfolding and aggregation of myoglobin, Biophys. J. 82 (2002) 2635–2644. [28] D.A. Prystupa, A.M. Donald, Infrared study of gelatin conformations in the gel and sol states, Polym. Gels Networks 4 (1996) 87–110. [29] R.K. Dukor, T.A. Keiderling, Mutarotation studies of poly-L-proline using FTIR, electronic and vibrational circular dichroism, Biospectroscopy 2 (1996) 83–100. [30] K.J. Payne, A. Veis, Fourier Transform IR spectroscopy of collagen and gelatin solutions: deconvolution of the amide I band for conformational studies, Biopolymers 27 (1988) 1749–1760. [31] W.F. Harrington, N.V. Rao, Collagen structure in solution. I. Kinetics of helix regeneration in single chain gelatin, Biochemistry 9 (1970) 3714–3724. [32] K. te Nijenhuis, Thermoreversible networks: viscoelastic properties and structure of gels, Advances in Polymer Science, Vol. 130, Springer, Berlin, 1997. [33] I. Noda, Y. Ozaki, Two-dimensional correlation spectroscopy—applications in vibrational and optical spectroscopy, Wiley, Chichester, UK, 2004. [34] Y.A. Lazarev, A.V. Lazareva, V.A. Shibnev, N.G. Esipova, Infrared spectra and structure of synthetic polypeptides, Biopolymers 17 (1978) 1197–1214. [35] J.H. Muyonga, C.G.B. Cole, K.G. Duodu, Fourier transform infrared (FTIR) spectroscopic study of acid soluble collagen and gelatin from skins and bones of young and adult Nile perch Lates niloticus, Food Chem. 86 (2004) 325–332. [36] J.P. Busnel, E.R. Morris, S.B. Ross-Murphy, Interpretation of the renaturation kinetics of gelatin solutions, Int. J. Biol. Macromol. 11 (1989) 119–125. [37] P.J. Flory, E.S.J. Weaver, Helix-coil transitions in dilute aqueous collagen solutions, Am. Chem. Soc. 82 (1960) 4518–4525. [38] J. Engel, Investigation of the denaturation and renaturation of soluble collagen by light scattering, Arch. Biochem. Biophys. 97 (1962) 150–158.