NMR and FTIR studies of hydrated pea proteins

Vol. 11 no. 4 pp. 485-491, 1997

Food Hydrocolloids

NMR and FTIR studies of hydrated pea proteins P.S.Belton, T.Bogracheva, Z.Cserhalmi 1, B.Czukor1, A.Grant, N.Lambert and N.Wellner Institute of Food Research, Norwich Laboratory, Norwich Research Park, Co1ney, Norwich NR4 7UA, UK and 'Central Food Research Institute (KEKI), 1022 Budapest, Herman Otto u.15, Hungary

Abstract The effects of increasing D20 hydration on the plasticization of vicilin, legumin and albumin fractions from peas were investigated using solid state 1H-NMR transverse relaxation techniques. Measurements showed increases on hydration in the T2 and intensity of the exponential component of the relaxation decay. However, a Gaussian (more rigid) component remained throughout the sample composition range. This behaviour contrasted with that observed in barley storage proteins and would indicate considerably less plasticization in legume proteins. In 2H-NMR transverse relaxation measurements ofa highly D20 hydrated sample over a large temperature range, vicilin was shown to be hydrophilic in nature. However, the observed absorption of water by vicilin was less than in the HMW subunits of wheat. FTIR spectra show little structural change in vicilin and legumin on hydration, in contrast to changes occurring in the cereal proteins. These difftrences in behaviour may be ascribed to differences between the globular structure of the legume proteins and the more linear structure of the cereal proteins.

Introduction Legume protein is a major commercial food protein ingredient imparting structural properties to the host food. Soya protein is by far the most widely used legume food protein, although alternative sources have regularly attracted much interest. Protein from peas is one such alternative. Grown regularly in N. America, China, Russia and the E.U., pea proteins are chemically and structurally very similar to those of soya. Rheological measurements of pea proteinates, e.g. water holding, emulsion stability, foaming capacity and thermal gelation (1-5), also indicate high food functionality. Legume protein in general comprises three fractionsalbumin (water-soluble metabolic proteins) (6,7), 7S and 11S globulins (storage proteins termed vicilin and legumin respectively in pea) (8-1O)-and the content and composition of these fractions can influence rheological properties. Optimizing the composition of legume seeds for food functionality either by plant breeding or flour processing during manufacture would provide the food industry with more effective ingredients. The connection between protein structure/composition and food functionality is still not fully clear and requires further elucidation. In the present preliminary study we have investigated the hydration properties, a key food functionality, of three pea protein fractions using solid state

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nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) techniques. This is the first report of the use of such spectroscopic methods on legume seed proteins. IH_ and 2H-NMR relaxation measurements of samples, exchanged and hydrated with deuterium oxide (D 20 ), were used to monitor changes in mobility of the protein and behaviour of the hydrating water (11), whilst FTIR was used in determining change in protein secondary structure (12,13). Such methods have been applied in a number of studies on proteins including collagen (14) and elastin (15), and have led to important new developments in the understanding of the dynamics of the barley C-hordein (16) and the high molecular weight (HMW) subunits of wheat (17,18).

Material and methods Samples Three protein fractions were prepared from each of two types of pea seeds which between them were known to be good sources of the fractions of interest, i.e, vicilin, legumin and albumin (19). These were a wild-type, round-seeded pea, containing high levels of legumin, and a wrinkle-seeded pea rich in albumin. Vicilin levels were similar in both.

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SOS-PAGE analysis of the isolated fractions revealed no significant difference between the two types of peas, although differential scanning calorimetry showed that the legumin from wrinkle-seeded peas was less thermally stable than that from round-seeded peas (20). Flour was prepared from peas fine milled on an Alpine pin mill (Augsburg). All three fractions were extracted from the non-defatted flour. In addition, a legumin fraction was extracted from hexane defatted flour. Flour (l00 g) was dispersed in 1 1 of 0.1 mol/dm! tricine buffer at pH 8.5, containing 0.5 mol/dm' NaCI and 0.01 mmol/dm! EOTA, and stirred for 1 h. The whole was centrifuged and ammonium sulphate (75% saturation) was added to the supernatant to precipitate the albumin, legumin and some vicilin. The material was centrifuged and the supernatant and pellet retained for further processing as follows. The supernatant

This was constituted solely of vicilin, which was recovered by the addition of further ammonium sulphate (99% saturation) followed by centrifugation to obtain a pellet which was dissolved in 50 mmol/dm! ammonium hydrogen carbonate at pH 8 and exhaustively dialysed against the same at 5°C. The pellet

This was suspended in 50 m mol/dm! sodium acetate at pH 5 and exhaustively dialysed against the same at 5°C. Under these conditions, most of the albumin is soluble and the vicilin and legumin totally insoluble. After dialyses the sample was centrifuged. The supernatant, containing the albumin fraction, was dialysed against carbonate solution as above. The pellet was dissolved in and dialysed against carbonate solution as above. This latter fraction, termed the legumin fraction, contained mainly legumin with small amounts of vicilin and albumin. All centrifugation was for 50 min at 23 000 g at 20°C. All fractions were freeze-dried and stored at + 1°C under vacuum. IH-NMR spectroscopy

A range of 0 20 hydrated proteins were prepared for use in IH-NMR experiments. Protein samples were left for 24 h at room temperature in excess 0 20 to H/O exchange before being freeze dried. Between 10 and 15 mg of freeze-dried protein was then 0 20 hydrated, either by storage for up to 1 week at room temperature in controlled humidity jars or, in the case of the higher hydration level samples, by direct addition of 0 20. Water uptake was determined gravimetrically and expressed on a weight to weight basis, i.e. (weight 020/total weight of sample) x 100. IH-NMR relaxation experiments using these samples were carried out' at 298 K on a Bruker MSL 100 spectrometer operating at 100.13 MHz. Samples were contained in 5 mm diameter NMR tubes. The 90° pulse length was 1 IlS and the receiver dead time lOllS. Longitudinal relaxation

measurements in the laboratory frame, characterized by time constants TJ, were made using the 180° - t - 90° pulse sequence with 64 t increments of 100 ms. Longitudinal relaxation measurements in the rotating frame (TIp) were made using spin locking times ranging from 1 to 500 ms and a field strength of 40 kHz. Longitudinal relaxation, which is exponential in nature, was analysed using a simple linear regression program (Quickfit) operating on the spectrometer or Tablecurve curve fitting software (version 3.10, Jandel Scientific) running on a PC. Transverse relaxation measurements (T2) were made from the free induction decay (FlO) with 2K data points and a dwell time of l us, Spectra were also recorded. Transverse relaxation resulting from constrained polymer systems, i.e, those that are not freely mobile in solution, will not be describable in terms of simple relaxation rates and detailed analysis is not possible. Transverse relaxation data was therefore characterized in the first instance by T2*, the time taken for the NMR signal to decay to lie of its original value. The observed differences in the transverse relaxation of the pea proteins were, however, further investigated by fitting the relaxation curves to a combination of a Gaussian and exponential decay using Tablecurve software. This must be regarded as a parameterization of the data rather than an expression of the underlying physics. 2H-NMR spectroscopy

The relaxation properties of the bulk 0 20, together with the contribution of exchangeable nuclei, were studied in a highly hydrated protein sample using 2H-NMR spectroscopy. A 100 mg sample of vicilin prepared from wrinkle-seeded pea flour was placed in a 5 mm o.d. NMR tube and hydrated with 1 g of 0 20 (90.9% wlw 0 20). The tube was sealed and the sample left for 15 h at room temperature. 2H transverse relaxation measurements (T2) were then made using a Carr-Purcell Meiboom-Gill (CPMG) sequence over the temperature range 288-363 K on a Bruker MSL 300 spectrometer at 46.05 MHz with 90° and 180° pulse lengths of 15 and 30 IlS respectively. Receiver dead time was 15 IlS and 2K data points were collected with a pulse spacing of 4ms.

FfIR spectroscopy FTIR spectra were recorded at room temperature on a BioRad FTS-60 spectrometer. Samples were fully hydrated using distilled H 20 and loaded into a MicroCircle ATR cell (SpectraTech) with a ZnSe crystal. Some 256 scans at 2 em:' resolution were co-added and referenced against the empty cell. The spectra of the hydrated proteins were obtained by subtracting a water spectrum recorded under identical conditions. The cell was then drained and the protein which was deposited on the crystal surface dried down under a stream of dry air. A spectrum of the dry protein was then measured.

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IH-NMR experiments The main purpose of the IH-NMR experiments was to monitor changes in the molecular mobility of the pea proteins upon hydration. This was achieved by the measurement of proton relaxation time constants T), TIp and T 2• In these D 20 exchanged and hydrated proteins, the IH-NMR signal arises solely from the non-exchangeable protons and relaxation will not be affected by spin-exchange processes with the deuterons during the relaxing period. T 1 relaxation processes are due to the high frequency (108 Hz) motions of the molecule, and several contributions to T 1 relaxation in proteins have been identified (21).These include methyl group re-orientation, segmental motion, side-chain motion and proline ring puckering. T I relaxation at 298 K in vicilin, legumin and albumin, hydrated over a maximum range of 0-62% D 20 , was single exponential in all cases and consistent with observed spin lattice behaviour in other proteins (16,18). T I values in dry proteins varied from -300 to 430ms, and the changes which occurred on hydration were similar in all cases. Figure 1 shows a representative sample of legumin preparations. Initially, T I increased with increasing hydration and reached a maximum at -15% D 20 . Above 15%, T I values decreased and minima occurred in samples containing in the region of 40% D 20. Some slight differences in T I relaxation measurements were seen between the proteins. T I values of vicilin tended to be higher than those of either legumin or albumin, but in general the changes in relaxation time across the hydration range used were small and indicated a broad distribution of correlation times. The observed initial increases in T I values on hydration at

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low D 20 levels were unexpected although similar results in other protein samples had been reported (H.Tang, personal communication; B.D.Moore, personal communication). Motion in the dry sample may be on the long correlation time side of a T 1 minimum. When water is added motion increases, shifting the T I minimum to a lower temperature and increasing the efficiency of relaxation (16). If the addition of a small amount of water shifts the minimum to a lower temperature without greatly increasing the efficiency of relaxation, an increase in T I could result if the dry sample was close to the T I minimum and the wet sample was shifted from this position to one significantly higher on the high temperature side. TIp, which is sensitive to motions with frequencies in the region of 105 Hz, was measured in vicilin and legumin fractions. Relaxation in the dry vicilin samples was single exponential, and TIp values were in the region of 6 ms (Fig. 2). Hydration levels of up to -30% D 20 had little effect, but at 40% and above relaxation became bi-exponential, with the

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appearance of a longer TIp component, the value of which increased with increasing hydration. At the highest hydration levels, this longer component accounted for -40% of the relaxation. Unlike vicilin, TIp relaxation in legumin samples from non-defatted flour was bi-exponential at all hydration levels. However, the shorter TIp component represented most of the population (80-90%). The apparent difference in TIp relaxation behaviour between the two proteins may be due to the high lipid content of the legumin fractions (20) since the analysis of TIp relaxation in legumin prepared from defatted flour was similar to that of the vicilin. I H spectra of dry, D 20 exchanged pea proteins consisted characteristically of a broad (more rigid) and a narrow (more mobile) component. There were, however, some slight differences in the shapes of spectra, with the sharpest peaks occurring in albumin spectra and the least sharp in legumin spectra. In general, as proteins are hydrated, there is a reduction in the amount of the broad component and corresponding increases in the narrow (Fig. 3). Spectra of dry legumin prepared from non-defatted flour showed much less of a broad component than vicilin or albumin, or indeed legumin prepared from defatted flour, and were not significantly changed on hydration. Non-defatted legumin spectra were dominated by a large peak, the intensity of which may in part be due to the presence of a highly mobile lipid component. Initially, the transverse relaxation properties of dry and hydrated protein fractions were compared using a single parameter, T2*, to characterize the decay. An increase in T2* value signifies a decrease in relaxation rate and an increase in

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protein mobility. In all protein fractions, T2* values increased with increasing hydration. In both vicilin and albumin there appeared a sharp transition in T2* between the 30 and 40% hydration levels, but in legumin, such transitions were less well defined (Fig. 4). These differences were further explored by fitting the experimental free induction decays (FIOs) as a sum of a Gaussian and exponential decay, bearing in mind the restrictions stated above. In general, the shape of the FlO changes from Gaussian to exponential with lengthening of the relaxation time as 0 20 is added, i.e. as the molecule becomes more mobile. In vicilin and albumin, the T2 value of both the Gaussian and exponential components increased on hydration, with more rapid increases occurring in the exponential component at hydration levels >40% 0 20 (Fig. 5). The populations of the exponential component in both proteins remained small (-10%) at levels <20% 0 20 but increased on further hydration, possibly reaching a maximum (40%) in vicilin samples hydrated to the 50% 0 20 level. The exponential component population of albumin continued to increase with increasing hydration, but the greater solubility of the albumin may influence results at the higher hydration levels. Differences were seen between the transverse relaxation behaviour of the legumin samples prepared from non-defatted and defatted flour. In dry legumin from non-defatted flour, the value of the exponential component was high (600-700ms) and tended to decrease with increases in hydration. In defatted legumin, exponential component values were much less, i.e, in the region of 100 ms, and increased with hydration (Fig. 6). In

Figure 6 The effect of hydration (D20) on 1H T2 in legumin fraction from defatted wrinkle-seeded pea flour. (A, B) absolute values and (C) percentage contributions of first (.) and second (D) components.

this respect, defatted legumin was similar to both vicilin and albumin. However, increases in the exponential population of both legumin samples on hydration were generally more linear than those of either vicilin or albumin. In the case of the vicilin and legumin samples, slight differences in relaxation measurements were seen between proteins from different sources. Relaxation rates tended to be lower in samples prepared from wrinkle-seeded peas than in similarly hydrated samples from round-seeded peas. In contrast, source appeared to have little effect on the relaxation behaviour of the albumin samples. 2H-NMR experiments 2H transverse relaxation (T2) measurements of a highly D 20 hydrated vicilin sample were made over the temperature range 283-363 K using a CPMG pulse sequence and compared with those of the HMW subunit of wheat (Fig. 7). In contrast to the protein lH transverse relaxation measurements above, the CPMG pulse sequence is used to measure the T2 (ms to s range) of more mobile species such as hydrating water. At 283 K, the T2 values of the components in the bi-exponential relaxation were -40 and 200 ms. In general, increased temperatures resulted in increased T 2 values, which in the case of the longer component peaked at 343 K. Populations of the longer T 2 component decreased from 30 to -10% with corresponding increases in the shorter T2 component. This relaxation behaviour may be typical of systems undergoing diffusive exchange (17) between different regions of the sample. In the vicilin sample studied here, the latter will be between the protein surface and the bulk

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water regions. The decrease in the proportion of the longer T2 component may be interpreted as the inclusion of water into the protein as the temperature was increased. This essentially hydrophilic behaviour has a common trend with that of the wheat gluten proteins and differs from that of elastin (15,17).

FUR experiments Comparison of the various protein fractions from pea flour showed that the FTIR spectra of vicilin and legumin were rather similar (Fig. 8A,B). In both, the amide I band peaks at 1630 cm! are due to a high content of f3-sheet structures. Hydration did not alter the shape of this band, in spite of a marked shift in the (unusually strong) amide II band from 1515 em:' to 1544 crrr l. The amide I band is a reliable

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indicator of secondary structures, whereas the amide II band is strongly affected by the binding of water molecules. Therefore this result means that the secondary structures of vici1in and legumin were not significantly changed by hydration. The albumin fraction from peas was clearly different (Fig. 8C). In the dry sample, the maximum of the amide I band at 1648 cm! indicated the presence of a high percentage of a-helix and unordered structures. The shoulder at 1636 crrr' showed some f3-sheet. On hydration, the amide I band was narrowed and the maximum shifted to 1637 crrr'. These changes are possibly due to the formation of more f3-sheet structure from unordered structures, whereas some a-helix content (shoulder at 1651 cm") apparently remained.

NMR and FTIR studies of hydrated pea proteins

Discussion The general effect of hydration on spin lattice r:l~xa~ion is small. The behaviour is consistent with the plasticization of the protein with water but no dramatic changes.in behaviour are observed. Similar effects have been seen in the barley protein C-hordein. The prob~ble cause o~ the insensitivity of spin lattice relaxation both m the rotatmg and laboratory frames is that the higher frequency motions, such as methyl group rotation and ring puckering, which ar~ ~es~ons~ble are relatively insensitive to water content. Plasticization IS more likely to affect slower backbone motions whic.h ar; better measured by transverse relaxation. The changes m T2 a~e of the same order as those in TIp' However, further analysis of the curves into Gaussian and exponential components shows the persistence of a Gaussian component throughout the composition range although its relative intensity chan~es. The exponential component increases in both T2 and .relatIve intensity. In contrast to this, the spectral behaviour of C-hordein (16) shows very little change in the narrow component but a total loss of the broad component. These are equivalent to the Gaussian and exponential components respectively. This indicates that the plasticization of the globular legume proteins is considerably less than that of the linear barley proteins. The absorption of water by the protein with increasing temperature indicates hydrophilic behaviour. However, the amount of water taken in is relatively little when compared with the HMW subunits of wheat gluten. Data are not available for C-hordein, but since it has been shown that C-hordein and the HMW subunits behave in a similar way (18) in their other NMR characteristics,. it may be assum~d that the HMW subunits are typical of linear cereal protem behaviour. The difference in behaviour may be ascribed to the differences in structure between the proteins. In linear cereal proteins hydration results in the f~rmati.on ?f large, ~pen, mainly unstructured loops (16). This option IS not available to legume proteins constrained in globular confo~mation~. Thus expansion of the protein is limited and water ingress IS curtailed. The infrared spectra, except in the case of albumin, support this view. Little secondary structural change is seen in vicilin and legumin, indicating that apart from some general hydration effects, water makes little difference to the protein. This is in contrast to the case in cereals. These differences in structural and dynamic responses go some way to explaining the differences observed in the functional behaviour of the proteins.

Acknowledgements This research is in part supported by the British Council in Budapest under the Cooperative Science and Technology Research and Development Programme (Joint BritishHungarian Cooperation). Funding by the UK Office of Science and Technology and the BBSRC is also gratefully

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acknowledged. The authors would also wish to thank Dr Cliff Hedley of the John Innes Research Institute, Norwich for the supply of pea seeds.

References 1. Sosulski,F.Wand McCurdy,A.R. (1987) 1. Food Sci., 52, 1010-1014. 2. Naczk,M., Rubin L.I and Shanhidi,F. (1986) 1. Food Sci., 51, 1245-1247. 3. Koyoro,H. and Powers,IR. (1987) Cereal Chem., 64, 97-101. 4. Dagorn-Scaviner,C., Gueguen,l and Lefebvre,l (1987) 1. Food Sci., 52, 335-341. 5. Wright,D.I and Bumstead,M.R. (1984) Phil. Trans. R Soc. Lond. B,304, 381-393. 6. Grant,D.R., Sumner,A.K. and Johnson,J. (1976) Can. Inst. Food Sci., Technol. 1. 9, 84-91. 7. Schroeder,H.E. (1984) 1. Sci. Food Agric., 35,191-198. 8. Wright,D.I (1987) In Hudson.BilE (ed.), Development in Food Proteins. Elsevier Applied Science, London, Vol. 5, pp.81-157. 9. Gatehouse,IA., Croy,R.R.D. and Boulter,D. (1984) CRC Crit. Rev. Plant Sciences, 1, 287-314. 10. Dieckert,IW and Dieckert,M.C. (1985) In Altschul,A.M. and Wilcke,H.L. (eds), New Protein Foods. Academic Press Inc., Orlando, Vol. 5, pp. 1-25. II. Belton,P.S. (1994) Prog. Biophys. Mol. Bioi., 61, 61-79. 12. Krimm,S. and Bandekar,l (1986) Adv. Protein Chem., 38, 181-364. 13. Careri,G., Giansanti,A. and Gratton,E. (1979) Biopolymers, 18, 1187-1203. 14. Edzes,H.T. and Samulski,E.T. (1978) 1. Magn. Reson., 31, 207-229. IS. Ellis,G.E. and Packer,K.I (1976) Biopolymers, 15, 813-832. 16. Belton,P.S., Gil,A.M. and Tatham,A.S. (1994) 1. Chem. Soc. Farad. Trans., 90,1099-1103. 17. Belton,P.S., Colquhoun.Ll., Field,IM., Grant,A., Shewry,P.R. and Tatham,A.S. (1994) 1. Cereal Sci., 19, 115-121. 18. Belton,P.S., Colquhoun,I.J., Field,IM., Grant,A., ShewrY,P.R., Tatham,A.S. and Wellner,N. (1995) Int. 1. Bioi. Macromol., 17, 74-80. 19. Wang,T.L. and Hedley,C.L. (1991) Seed Sci. Res., 1, 1-14. 20. Perez, M D., Chambers,S.I, Bacon,IR., Lambert,N., Hedley,C.L. and Wang,T.L. (1993) Seed Sci. Res., 3, 187-194. 21. Andrew,E.R. (1985) Polymer, 26, 190-192. Received on September 14, 1996; accepted on December 4, 1996