低离子强度下调节pH值对鲤肌球蛋白空间构象的影响
|
摘要
探讨了低离子强度(0.05 mol/L KCl)条件下,调节pH对鲤肌球蛋白空间构象的影响。试验结果表明:肌球蛋白于酸性pH(2.5,3.5)或碱性pH(11.5,12.5)处理后,溶解度均达到90%以上。pH调节处理后,肌球蛋白Ca~(2+)-ATPase完全失活,即使调回中性也无法恢复,说明肌球蛋白头部结构遭到破坏。圆二色谱分析发现α-螺旋含量没有变化,表明pH调节处理对肌球蛋白尾部二级结构没有影响。此外,经不同pH条件下的酸、碱处理,肌球蛋白的色氨酸荧光发生淬灭,表面疏水性升高,巯基含量降低,胰凝乳蛋白酶酶切位点发生改变,这些都表明肌球蛋白的三级结构遭到破坏。
In this study, the effect of pH-shift on the conformation of myosin from common carp at low ionic strength was investigated. The results showed that the solubility of the myosin was over 90% at low ionic strength(0.05 mol/L KCl) and extreme acid-Alkali conditions(pH 2.5, pH 3.5, pH 11.5 and pH 12.5). After pH treatment, the Ca~(2+)-ATPase in myosin was inactivated completely, and the recovery was impracticable even if at neutral pH again, it suggested that the head structure of myosin was already devastated. Circular dichroismspectroscopy analysis showed that the content of α-helix scarcely changed, which indicated that pH treatment had no effect on the tail structure of myosin.And we also found that after the process of different pH treatment, the tryptophan fluorescence of myosin was quenched,the surface hydrophobicity was increased, the sulfhydryl content was decreased and a change was induced at chymotrypsin cleavage sites, which indicated that the tertiary structure of myosin was changed.
In this study, the effect of pH-shift on the conformation of myosin from common carp at low ionic strength was investigated. The results showed that the solubility of the myosin was over 90% at low ionic strength(0.05 mol/L KCl) and extreme acid-Alkali conditions(pH 2.5, pH 3.5, pH 11.5 and pH 12.5). After pH treatment, the Ca~(2+)-ATPase in myosin was inactivated completely, and the recovery was impracticable even if at neutral pH again, it suggested that the head structure of myosin was already devastated. Circular dichroismspectroscopy analysis showed that the content of α-helix scarcely changed, which indicated that pH treatment had no effect on the tail structure of myosin.And we also found that after the process of different pH treatment, the tryptophan fluorescence of myosin was quenched,the surface hydrophobicity was increased, the sulfhydryl content was decreased and a change was induced at chymotrypsin cleavage sites, which indicated that the tertiary structure of myosin was changed.
引文
[1]农业部渔业局. 2015年中国渔业统计年鉴[M].北京:中国农业出版社, 2015:30-31.
[2]MARTIN-SANCHEZ A M, NAVARRO C, PEREZALVAREZ J A, et al. Alternatives for efficient and sustainable production of surimi:a review[J]. Comprehensive Reviews in Food Science and Food Safety, 2009, 8(4):359-374.
[3]WANG H N, WU J, BETTI M. Chemical, rheological and surface morphologic characterisation of spent hen proteins extracted by ph-shift processing with or without the presence of cryoprotectants[J].Food Chemistry, 2013, 139(1/4):710-719.
[4]OMANA D A. Alkali-aided protein extraction from chicken dark meat:textural properties and color characteristics of recovered proteins[J]. Poultry Science, 2010, 89(5):1056-1064.
[5]CAVONIUS L R, ALBERS E, UNDELAND I. pHshift processing of nannochloropsis oculata, microalgal biomass to obtain a protein-enriched food or feed ingredient[J]. Algal Research, 2015, 5(22):95-102.
[6]OTTO R A, BEAMER S, JACZYNSKI J, et al.The effect of using citric or acetic acid on survival of Listeria monocytogenes, during fish protein recovery by isoelectric solubilization and precipitation process[J]. Journal of Food Science, 2011, 76(8):579-583.
[7]XU Y, XIA W, JIANG Q, et al. Acid-induced aggregation of actomyosinfrom silver carp(Hypophthalmichthys molitrix)[J]. Food Hydrocolloids, 2012,27(2):309-315.
[8]PAKER I, BEAMER S, JACZYNSKI J, et al. pH shift protein recovery with organic acids on texture and color of cooked gels[J]. Journal of the Science of Food&Agriculture, 2015, 95(2):275-280.
[9]TIAN Y, WANG W, YUAN C, et al. Nutritional and digestive properties of protein isolates extracted from the muscle of the common carp using ph-shift processing[J]. Journal of Food Processing&Preservation, 2017, 41(1):e12847.
[10]KRISTINSSON H G, HULTIN H O. Changes in conformation and subunit assembly of cod myosin at low and high pH and after subsequent refolding[J].Journal of Agricultural&Food Chemistry, 2003, 51(24):7187-7196.
[11]KATO S, KONNO K. Isolation of carp myosin rod and its structural stability[J]. Nihon-suisan-gakkaishi, 1993, 59(3):539-544.
[12]THAVAROJ W, SAKAMOTO M, KONNO Y, et al.Preceding actin denaturation accelerates myosin denaturation in tilapia myofibrils in frozen storage[J].Fisheries Science, 2016, 82(5):843-850.
[13]BENJAKUL S, SEYMOUR T A, MORRISSEY M T, et al. Physicochemical changes in pacific whiting muscle proteins during iced storage[J]. Journal of Food Science, 1997, 62(4):729-733.
[14]CHAU H F, WAN K Y, YAN K K, et al. Methods of testing protein functionality[M]. Methods of Testing Protein Functionality. 1996.
[15]TADPITCHAYANGKOON P, PARK J W, YONGSAWATDIGUL J. Conformational changes and dynamic rheological properties of fish sarcoplasmic proteins treated at various pHs[J]. Food Chemistry,2010, 121(4):1046-1052.
[16]HAMM R. Biochemistry of meat hydration[J]. Advances in Food Research, 1960, 10(10):355-463.
[17]STEFANSSON G, HULTIN H O. On the solubility of cod muscle proteins in water[J]. Journal of Agri cultural&Food Chemistry, 2002, 42(12):2656-2664.
[18]AND T M L, PARK J W. Solubility of salmon myosin as affected by conformational changes at various ionic strengths and pH[J]. Journal of Food Science, 1998, 63(2):215-218.
[19]YONGSAWATDIGUL J, PARK J W. Effects of alkali and acid solubilization on gelation characteristics of rockfish muscle proteins[J]. Journal of Food Science, 2004, 69(7):499-505.
[20]THAWORNCHINSOMBUT S, PARK J W. Role of ph in solubility and conformational changes of pacific whiting muscle proteins[J]. Journal of Food Biochemistry, 2004, 28(28):135-154.
[21]SHARP A, GERALD OFFER. The mechanism of formation of gels from myosin molecules[J]. Journal of the Science of Food&Agriculture, 1992, 58(1):63-73.
[22]RAGHAVAN S, KRISTINSSON H G. Conformational and rheological changes in catfish myosin during alkali-induced unfolding and refolding[J]. Food Chemistry, 2008, 107(1):385-398.
[23]KIM Y S, PARK J W, CHOI Y J. New approaches for the effective recovery of fish proteins and their physicochemical characteristics[J]. Fisheries Science,2003, 69(6):1231-1239.
[24]HAMM R. Biochemistry of meat hydration[J]. Advances in Food Research, 1960, 10(10):355-463.
[2]MARTIN-SANCHEZ A M, NAVARRO C, PEREZALVAREZ J A, et al. Alternatives for efficient and sustainable production of surimi:a review[J]. Comprehensive Reviews in Food Science and Food Safety, 2009, 8(4):359-374.
[3]WANG H N, WU J, BETTI M. Chemical, rheological and surface morphologic characterisation of spent hen proteins extracted by ph-shift processing with or without the presence of cryoprotectants[J].Food Chemistry, 2013, 139(1/4):710-719.
[4]OMANA D A. Alkali-aided protein extraction from chicken dark meat:textural properties and color characteristics of recovered proteins[J]. Poultry Science, 2010, 89(5):1056-1064.
[5]CAVONIUS L R, ALBERS E, UNDELAND I. pHshift processing of nannochloropsis oculata, microalgal biomass to obtain a protein-enriched food or feed ingredient[J]. Algal Research, 2015, 5(22):95-102.
[6]OTTO R A, BEAMER S, JACZYNSKI J, et al.The effect of using citric or acetic acid on survival of Listeria monocytogenes, during fish protein recovery by isoelectric solubilization and precipitation process[J]. Journal of Food Science, 2011, 76(8):579-583.
[7]XU Y, XIA W, JIANG Q, et al. Acid-induced aggregation of actomyosinfrom silver carp(Hypophthalmichthys molitrix)[J]. Food Hydrocolloids, 2012,27(2):309-315.
[8]PAKER I, BEAMER S, JACZYNSKI J, et al. pH shift protein recovery with organic acids on texture and color of cooked gels[J]. Journal of the Science of Food&Agriculture, 2015, 95(2):275-280.
[9]TIAN Y, WANG W, YUAN C, et al. Nutritional and digestive properties of protein isolates extracted from the muscle of the common carp using ph-shift processing[J]. Journal of Food Processing&Preservation, 2017, 41(1):e12847.
[10]KRISTINSSON H G, HULTIN H O. Changes in conformation and subunit assembly of cod myosin at low and high pH and after subsequent refolding[J].Journal of Agricultural&Food Chemistry, 2003, 51(24):7187-7196.
[11]KATO S, KONNO K. Isolation of carp myosin rod and its structural stability[J]. Nihon-suisan-gakkaishi, 1993, 59(3):539-544.
[12]THAVAROJ W, SAKAMOTO M, KONNO Y, et al.Preceding actin denaturation accelerates myosin denaturation in tilapia myofibrils in frozen storage[J].Fisheries Science, 2016, 82(5):843-850.
[13]BENJAKUL S, SEYMOUR T A, MORRISSEY M T, et al. Physicochemical changes in pacific whiting muscle proteins during iced storage[J]. Journal of Food Science, 1997, 62(4):729-733.
[14]CHAU H F, WAN K Y, YAN K K, et al. Methods of testing protein functionality[M]. Methods of Testing Protein Functionality. 1996.
[15]TADPITCHAYANGKOON P, PARK J W, YONGSAWATDIGUL J. Conformational changes and dynamic rheological properties of fish sarcoplasmic proteins treated at various pHs[J]. Food Chemistry,2010, 121(4):1046-1052.
[16]HAMM R. Biochemistry of meat hydration[J]. Advances in Food Research, 1960, 10(10):355-463.
[17]STEFANSSON G, HULTIN H O. On the solubility of cod muscle proteins in water[J]. Journal of Agri cultural&Food Chemistry, 2002, 42(12):2656-2664.
[18]AND T M L, PARK J W. Solubility of salmon myosin as affected by conformational changes at various ionic strengths and pH[J]. Journal of Food Science, 1998, 63(2):215-218.
[19]YONGSAWATDIGUL J, PARK J W. Effects of alkali and acid solubilization on gelation characteristics of rockfish muscle proteins[J]. Journal of Food Science, 2004, 69(7):499-505.
[20]THAWORNCHINSOMBUT S, PARK J W. Role of ph in solubility and conformational changes of pacific whiting muscle proteins[J]. Journal of Food Biochemistry, 2004, 28(28):135-154.
[21]SHARP A, GERALD OFFER. The mechanism of formation of gels from myosin molecules[J]. Journal of the Science of Food&Agriculture, 1992, 58(1):63-73.
[22]RAGHAVAN S, KRISTINSSON H G. Conformational and rheological changes in catfish myosin during alkali-induced unfolding and refolding[J]. Food Chemistry, 2008, 107(1):385-398.
[23]KIM Y S, PARK J W, CHOI Y J. New approaches for the effective recovery of fish proteins and their physicochemical characteristics[J]. Fisheries Science,2003, 69(6):1231-1239.
[24]HAMM R. Biochemistry of meat hydration[J]. Advances in Food Research, 1960, 10(10):355-463.