Heat Denaturation and Emulsifying Properties of Plasma Protein

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1 Agric. Biol. Chem., 51 (10), , Heat Denaturation and Emulsifying Properties of Plasma Protein Masayoshi Saito and Harue Taira National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Yatabe-machi, Tsukuba-gun, Ibaraki 305, Japan Received May 27, 1987 The effects of ph and NaCl on the denaturation of plasma protein during heat treatment were investigated, as well as the relationship between protein structure and emulsifying properties. When the plasma protein solution (1% w/v) was heated at 80 C, precipitation was accelerated by the presence of NaCl. The measurement of SH groups, surface hydrophobicity and CDspectrum revealed that denaturation occurs easily by heat treatment in the neutral ph region and in the presence ofnacl. The emulsifying activity index (EAI) did not change much after heat treatment at ph 3 irrespective of the presence ofnacl, but it decreased about 60% after heat treatment at ph 7 in the absence of NaCl. Gel filtration patterns indicated that a high molecular weight peak arose upon heat treatment at neutral ph. Weconcluded that the decrease in EAI was owing to the polymerization of serum albumin and y-globulin, which are the main components of plasma protein, and disulflde bonds participated in this process. Animal blood collected at slaughterhouses is a potential source of dietary proteins, but most of it has been wasted. In recent years, interest in the use of blood protein as a food material has been increasing, which necessitates the development of a hygienic blood collection system in slaughterhouses. Plasma proteins have been reported to have good functional properties such as gelation and emulsification,1'2) so their use in food processing has a lot of advantages. However, the functional properties have complex features and most of them are still obscure, and the use of plasma proteins is limited to an additive in meat products such as hamand sausage at present. It is necessary to understand their functional properties and find the best uses for their functional properties. In Japan, plasma which is to be used in food processing must be sterilized by heat treatment, although this causes a serious denaturation of plasma proteins. At a high concentration of plasma protein solution (above 6%), its viscosity increases and it forms a gel during heat treatment.3'4) In our previous report,5) we found the best emulsifying properties of plasma protein at 1%to 2%. However, the heat denaturation of plasma proteins in solution at such a low concentration has not been studied. In this study the effects of ph and NaCl on the denaturation and the emulsifying properties of 1%plasma protein solution during heat treatment were investigated, as well as the relationship between protein structure and emulsifying properties. MATERIALS AND METHODS Preparation ofplasma protein. Whole porcine blood was obtained from the National Institute of Animal Industry, Tsukuba. Blood was collected at the time of slaughter and an sodium citrate solution (5% w/v) was added as an anticoagulant at a level of 100ml/1 of blood. The mixture was immediately cooled to 4 C in ail ice bath, and centrifuged at 1600 x g at 4 C for 15 min. The supernatant was further centrifuged at 6400xg at 4 C for 15min, dialysed against deionized water at 4 C, and freeze-dried. Heat treatment and turbidimetry. The plasma protein powder was dissolved in water or 0.2m NaCl solution (1% w/v) and itsphwasadjusted at 3 or 7with 1 nhc1 or 1n NaOH. Three milliliters of the protein solution in a glass tube (12x 105mm) was incubated at 80 C with shaking.

2788 M. Saito and H. Taira After the heat treatment, the solution was immediately cooled in a water bath to room temperature, and the turbidity of the protein suspension was measured at 600nm.

2 2788 M. Saito and H. Taira After the heat treatment, the solution was immediately cooled in a water bath to room temperature, and the turbidity of the protein suspension was measured at 600nm. Measurement of sulfhydryl groups. Sulfhydryl groups were measured by a modification of the procedure of Ellman.6) A solution of 0. 1 ml of 5,5-dithiobis (2-nitrobenzenoic acid) (DTNB) (40mg of DTNB in 10ml of 0.1m phosphate buffer, ph 7.0) was added to the mixture of 1 ml of protein solution (1% w/v), 4ml of0.1% sodium dodecyl sulfate (SDS) in 0.1 m phosphate buffer (ph 8.0), and 5ml of 0.1m phosphate buffer (ph 8.0). Following the incubation for 30min at room temperature, its absorbance was measured at 412nm. A molecular extinction coefficient of 1.36x 104/m cmwas used to calculate moles of sulfhydryl groups per gram of protein. Measurement of hydrophobicity. The surface hydrophobicity of proteins was measured by the fluorescence probe method using ds-parinaric acid, by the procedure of Kato and Nakai,7) except that the protein was dissolved in 0.01 m phosphate buffer (ph 7.0) containing 0.002% SDS. Measurement of circular dichroism (CD). A JASCO J- 500Aspectropolarimeter was used to record CDspcetra. The protein dispersion was centrifuged at 25000xg at 20 C for 20 min, then diluted to the protein concentration of 0.037% with 0.05m phosphate buffer (ph 7.0). The molar ellipticity per residue at 222nm ([^222) was calculated and used as the index of protein denaturation. Measurement of emulsifying activity. The emulsifying activity index (EAI) was measured by the turbidimetric procedure of Pearce and Kinsella.8) Two milliliters of protein solution (1% w/v) and 0.5g of soybean oil were homogenized at 30 C for 3min by a Polytron PT-7 (Kinematica, Switzerland) at its maximum speed. A sample (0.5ml) of the emulsion was diluted with a 0.1% SDSsolution and its turbidity was measured at 500nm. High performance liquid chromatography (HPLC). The samples were analyzed with a JASCOTrirotar III High Performance Liquid Chromatograph with a TSK gel G3000 SWcolumn (7.5 x 600mm) and a UV detector at a wavelength of 280nm. The sample solution was eluted with 0.2m phosphate buffer (ph 7.2) containing 0.05% NaN3at room temperature The molecular weight at a flow rate was measured of 0.5ml/min. using a Low Molecular Weight Calibration Kit (Pharmacia Fine Chemicals). Gel filtration chromatography. Gel filtration was done with a Sephadex G-100 column (26 x 900mm) by eluting the sample with 0.2m phosphate buffer (ph 7.2) containing 0.05% NaN3 at 4 C at a flow rate of 30ml/hr. The absorbance of the fractions was measured at 28nm. The fractions were dialysed against deionized water at 4 C and concentrated to one-third or one-tenth of its original volume with a MINICON-B15concentrator (Amicon). Protein in the concentrated samples was measured by the procedure of Lowry et al.9) Sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE). SDS-PAGEwas done by. the procedure of Laemmli10) using a 8% acrylamide separation gel. After electrophoresis, the gels were stained with Coomassie blue R-250. Each plasma protein was identified by comparing its electrophoretic mobility with those of the standard proteins purchased from Sigma Chemical Co. RESULTS AND DISCUSSION Coagulation of plasma protein by heat treatment Coagulation during heat treatment was monitored by the turbidity at 600nm. Figure 1 shows the change in the turbidity of 1% (w/v) plasma protein at 80 C. At ph 7 in water, the turbidity of the heated sample remained at a low level, while at ph 7 in 0.2m NaCl the turbidity increased rapidly during heat treatment and reached a plateau within 30min. At ph 3 in water the turbidity was very low,- but at ph 3 in 0.2m NaCl it increased slightly during heat treatment. Shimada and Matsushita11} have reported that bovine serum albumin (BSA) which is the main component of plasma protein showed a rapid thermal coagulation in the presence of 0.3mNaClfrompH 5 to ll. The sameeffect of NaCl on thermal coagulation was observed with the plasma protein in this work. These results indicate that heat treatment of plasma protein should be done in the absence ofnacl or at an acidic ph (ph 3) to avoid coagulation. Denaturation of plasma protein by heat treatment The degree of the denaturation of protein is generally judged from the amount of sulfhydryl groups, surface hydrophobicity, and CDspectrum. Table I shows the amount of sulfhydryl groups and surface hydrophobicity of the heat-treated samples. At ph 7 with 0.2m NaCl, they could not be measured because of

3 Heat Denaturation of Plasma Protein D600 [9]222x10 3deg cm2/dmol *v -9 ^ à" Heating time min Fig. 1. Change in Turbidity of Plasma Protein Solution during Heat Treatment. Protein solution (1% w/v) was heated at 80 C. #, at ph 7 inwater and atph 3 inwater; A, atph 7 in 0.2mNaCl; O,atpH 3 ino.2mnacl. Table I. Amount of SH Groups and Hydrophobicity of Heat-treated Plasma Protein Heat treatment was at 80 C for 30min. a Relative values. precipitation. The amount of sulfhydryl groups of plasma protein was decreased by the heat treatment at ph 7 in water, which suggests the formation of disulflde bonds or damage to the sulfhydryl groups. The amount of sulfhydryl groups was not altered by the heat treatment at ph 3 either in water or in 0.2m NaCl. These observations agree with the fact that sulfhydryl groups dissociate and react more activily at ph 7 than at ph 3. The relationship between protein surface hydrophobicity and protein structure or functional properties has been reported.7' 12) From Table I, the surface hydrophobicity of plasma protein was increased by heat. This effect was more outstanding at ph 7 in water and atph Heating time min Fig. 2. Change in Molar Ellipticity per Residue at 222 nm of Plasma Protein during Heat Treatment. Protein solution (1% w/v) was heated at 80 C and diluted to 0.037%. #, atph 7inwater; A, atph 3 inwater. in 0.2m NaCl. The increase in surface hydrophobicity was small at ph 3 in water. This result suggests that the surface hydrophobicity of the plasma protein is easy to increase and denaturation occurs easily during the heat treatment at neutral ph (ph 7) and the presence of 0.2m NaCl. Changes in the ellipticity at 222nm ([0]222) have been used as a parameter for protein denaturation, because it decreases with damage to the ordered protein structure. Figure 2 shows the change in [6]222 during the heat treatment at 80 C. CDcould not be measured in 0.2m NaCl, because of precipitation. [0]222 decreased more rapidly at ph 7 than at ph 3, which also suggests that denaturation of the plasma protein during the heat treatment occurs more easily at ph 7 than at ph 3. Changesin emulsifying properties upon heat treatment It has been reported that plasma protein is an excellent emulsifier,5'13) but the changes in its properties during heat treatment are still obscure. Figure 3 shows the change in EAI during heat treatment at 80 C. EAI decreased greatly during the heat treatment at ph 7 in water, though this protein solution showed no increase in the turbidity. At ph 3 either in water or in 0.2m NaCl, the EAI decreased with the time of heat treatment. After 4min, it was observed that EAI gradually increased and

4 2790 M. Saito and H. Taira mvg OD28o c Heating 30 min time Fig. 3. Change in Emulsifying Activity Index of Plasma Protein during Heat Treatment. Protein solution (1% w/v) was heated at 80 C. A, at ph 7inwater; A,atpH 7in0.2mNaCl; +, atph 3 inwater; O,atpH 3in0.2mNaCl. after 10 min they reached almost the same level as that of the untreated sample. As ph 7 in 0.2m NaCI, EAI was increased by heat treatment for 2 min, then it gradually decreased and reached the same level as that of the untreated sample and remained constant. Howeverthese samples contained much precipitation, so this does not simply suggest that these samples have the same emulsifying activity as the untreated sample. A significant correlation has been observed between EAI and surface hydrophobicity in manynative and denatured proteins.7) Because plasma protein is a mixture of manyproteins, the change in hydrophobicity is a complex phenomenon in this case. Surface hydrophobicity increased during the heat treatment (Table I), but EAI did not increase in this study. Especially in the heat treatment at ph 7 in water, surface hydrophobicity increased by about 70% after the heat treatment, but EAI decreased about 50%at the same time. This suggests that some other factors have a profound effect on EAI. La Retention time min Fig. 4. HPLCPatterns of Heat-treated Plasma Protein. Heat treatment was at 80 C for 30min. A, control (untreated); B,at ph 3 in water; C, at ph 7 in water. Hea t- induced polymerization In this section we investigated the mechanism of decrease in the EAI of plasma protein during heat treatment at ph 7 in water at 80 C, when no precipitation occurred. The amount of sulfhydryl groups of the plasma protein was decreased by the heat treatment, be formed which suggests that by disulfide bonds. polymers could A change in molecular weight was checked by HPLCgel filtration. Figure 4 shows the HPLCpatterns of the plasma proteins with or without heat treatment. With heat treatment at ph 7, the high molecular weight peak (peak H) increased greatly, but this peak did not increase so much in the sample heated at ph 3. This suggests that there is a relationship between the formation of the polymers and the decrease in EAI. To investigate the emulsifying properties and the composition of these polymers, the heat treated proteins were fractionated, and the polymer fraction was examined by SDS- PAGE or EAI measurement. The fractionation was done by gel filtration using a Sephadex G-100 column.

5 Heat Denaturation of Plasma Protein tod280.-, 2 I / HMWX/lMWX^ Elution volume m' Fig. 5. Gel Filtration Patterns of Heat-treated Plasma Protein. Heat treatment was at ph 7 in water at 80 C for 30min. 7Gf A B C Fig. 6. SDS-PAGEPatterns of Heat-treated and Fractionated Plasma Protein. Protein solution (1% w/v) was heated at 80 C for 30min, then fractionated by gel filtration. A, control (untreated whole plasma protein); B, HMW fraction; C, LMW fraction; SA, serum alubumin; yg, y-globulin (heavychain). Figure 5 shows the gel filtration chromatogram of the plasma protein heat-treated at ph 7 in water. The sample was separated into two fractions; a high molecular weight fraction (fraction HMW)and a low molecular weight fraction (fraction LMW). Figure 6 shows the SDS-PAGEpatterns of these fractions. The composition of the fraction HMWwas almost the same as that of the Table II. Emulsifying Activity Index of Whole and Fractionated Plasma Protein Heat treatment was at 80 C for 30min. whole plasma protein, and the main components were serum albumin and y-globulin. The main component of the fraction LMW was serum albumin and the amount of the other proteins in this fraction was small. In this case samples were reduced with 2-mercaptoethanol before electrophoresis and the fraction HMWwas dissociated to its original components. This suggests that disulfide bonds participate in the formation of fraction HMW, which is almost same composition as plasma protein. Table II shows the EAI of these fractions. The EAI of the fraction HMWwas smaller than that of the untreated sample and almost the same as that of the heat-treated sample, and the EAI of the fraction LMWwas almost the same as that of the untreated sample. These suggest that during the heat treatment proteins becomepolymers whose EAI is lower than that of the original proteins, and as a result, EAI of the whole heat-treated sample decreased. It can be concluded that the decrease of EAI during the heat treatment is due to the formation of polymers, although further investigation must be done to clarify the effects of protein structure on the decrease in EAI. Acknowledgments. The authors are grateful to Mr. H. Nakai of the National Institute of Animal Industry for providing the porcine blood, and Professor K. Yamauchi, Dr. S. Kaminogawa, and Dr. M. Shimizu of the Department of Agricultural Chemistry, the Univarsity of Tokyo for their helpful suggestions. REFERENCES 1) M. D. Ranken, "Applied Protein Chemistry," ed. by

6 2792 M. Saito and H. Taira R. A. Grant, Appl. Sci. Pub., London, 1980, p J. Wismer-Pedersen, Food Tech., 30, 76 (1979). J. P. Harper, D. A. Suter, C. W. DillandE. R. Jones, J. Food Sci., 43, 1204 (1978). N. Nakamura, Y. Yoshino, S. Inoue and T. Nagai, J. Jpn. Soc. Food Sci. Tech., 31, 454 (1984). M. Saito, M. Shimizu and K. Yamauchi, /. Jpn. Soc. Food Sci. Tech., 34, 223 (1987). G. L. Ellman, Arch. Biochem. Biophys., 82, 70 (1959). A. Kato and S. Nakai, Biochim. Biophys. Ada, 624, 13 (1980). K. N. Pearce and J. E. Kinsella, J. Agric. Food Chern., 26, 716 (1978). O. H. Lowry, N. J. Rosebrough,A. L. Farr and R. J. Randall, /. Biol. Chem., 193, 265 (1951). U. K. Laemmli, Nature, 227, 680 (1970). K. Shimada and S. Matsushita, Agric. Biol. Chem., 45, 1945 (1981). S. Nakai, J. Agric. Food Chem., 31, 676 (1983). P. T. Tybor, C. W. Dill and W. A. Landmann, J. FoodScL, 40, 155 (1975).

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