福林酚试剂法与缩二脲试剂法测定饲用蛋白酶活力的比较分析
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摘要
本试验采用福林酚试剂法和缩二脲试剂法分别测定10种饲用蛋白酶活力,并比较两种方法测得牛血清蛋白(BSA)和酪氨酸(Tyr)标准曲线的线性相关性和两种方法测定蛋白酶活力的准确性、灵敏度及变异度。结果表明,两种方法测得的两种标准曲线的线性相关性均较好,同时两种方法测得蛋白酶活力的精确度及变异度也均较好;相对于缩二脲试剂法,福林酚试剂法测定蛋白酶活力的灵敏度较高,检测限较低,同时福林酚试剂法测定中性或碱性蛋白酶活力准确性较高,但测定酸性蛋白酶活力准确性较低。普通氨基酸含量的测定可以选用福林酚试剂法,而双缩脲试剂法可以很好地测定饲料中蛋白质含量,故在实际工作中应根据需要选择合适的方法。
In this study,the protolytic activity of 10 feed proteases was measured by the Folinphenol reagent method and the Biuret reagent method respectively.Then,the linear correlation of standard curves of BSA and Tyr,and the accuracy,sensibility and variation of two methods were compared.The results showed that there were satisfactory linear correlations between the activity and the absorbance read using the two methods.The precision and variation of the enzyme preparations by the two methods were also acceptable.Compared with the Biuret reagent method,the Folinphenol reagent method produced the higher sensibility and lower detectability of enzyme activity,and more accurate measurement of neutral or alkaline proteases activities.However,the measurement of acid protease activity was less accurate using the Folinphenol reagent method.The content of amino acids could be measured by the flinol reagent method,while the content of protein in feedstuff could be measured by the biuret reagent method.The present study implied that we should select the appropriate method to measure the activity of proteases according to specific need in practical work.
In this study,the protolytic activity of 10 feed proteases was measured by the Folinphenol reagent method and the Biuret reagent method respectively.Then,the linear correlation of standard curves of BSA and Tyr,and the accuracy,sensibility and variation of two methods were compared.The results showed that there were satisfactory linear correlations between the activity and the absorbance read using the two methods.The precision and variation of the enzyme preparations by the two methods were also acceptable.Compared with the Biuret reagent method,the Folinphenol reagent method produced the higher sensibility and lower detectability of enzyme activity,and more accurate measurement of neutral or alkaline proteases activities.However,the measurement of acid protease activity was less accurate using the Folinphenol reagent method.The content of amino acids could be measured by the flinol reagent method,while the content of protein in feedstuff could be measured by the biuret reagent method.The present study implied that we should select the appropriate method to measure the activity of proteases according to specific need in practical work.
引文
[1]张桂凤,黄志勇,徐帅.饲用酶制剂的作用机理以及在畜禽生产中的应用效果[J].中国饲料添加剂,2009(11):10-13.
[2]李存富.关于蛋白质测定中几个问题的理论分析与讨论[J].中国酿造,2004,23(9):29-30.
[3]Friedman M.Applications of the ninhydrin reaction for analysis of amino acids,peptides,and proteins to agricultural and biomedical sciences[J].Journal of Agricultural&Food Chemistry,2004,52(3):385-406.
[4]Habeeb A F S A.Determination of free amino groups in proteins by trinitrobenzenesulfonic acid[J].Analytical Biochemistry,1966,14(3):328-336.
[5]Robbins R J,Bean S R.Development of a quantitative high-performance liquid chromatography-photodiode array detection measurement system for phenolic acids[J].Journal of Chromatography A,2004,1038(1):97-105.
[6]赵晓娟,李敏仪,黄桂颖,等.Folin-Ciocalteu法测定苹果醋饮料的总多酚含量[J].食品科学,2013,34(08):31-35.
[7]饲料添加剂酸性、中性蛋白酶活力的测定分光光度法:GB/T28715-2012[S].北京:中国标准出版社,2012.
[8]蛋白酶制剂:GB/T 23527-2009[S].北京:中国标准出版社,2009.
[9]Lubran M M.The measurement of total serum proteins by the Biuret method[J].Annals of Clinical&Laboratory Science,1978,8(2):106.
[10]Janairo G,Linley M,Yap L,et al.Determination of the Sensitivity Range of Biuret Test for Undergraduate Biochemistry Experiments[J].e-Journal of Science&Technology,2011(5):81-87.
[11]赵书景.粮食、饲料中蛋白质含量测定方法的研究[D].扬州:扬州大学,2008.
[12]Agbor G A,Vinson J A,Donnelly P E.Folin-Ciocalteau Reagent for Polyphenolic Assay[J].International Journal of Food Science Nutrition&Dietetics,2014,3(8),147-156.
[13]韩葵葵.黄酮类药物与血清蛋白的相互作用以及不同表面活性剂的影响[D].扬州:扬州大学,2008.
[14]Enechi O.C,Nwabueze Emilia C.A New Colorimetric Method for the Determination of Proteins[J].Advances in Biological Research,2013,7(5):159-162.
[15]Gornall A G,Bardawill C J,David M M.Determination of serum proteins by means of the biuret reaction[J].Journal of Biological Chemistry,1949,177(2):751-766.
[16]Daniel R M,Danson M J,Eisenthal R,et al.A new parameters controlling the effect of temperature on enzyme activity[J].Biochemical Society Transactions,2007,35(6):1536-1543.
[17]Fromm H J.The Effect of Temperature and p H on Enzyme Activity[M].Initial Rate Enzyme Kinetics.Springer Berlin Heidelberg,1975:201-235.
[18]Williams A.Mechanism of action and specificity of proteolytic enzymes[J].Quarterly Reviews Chemical Society,1969,23(1):1-17.
[19]Raghunath M,Rao B N.Determination of dipeptides in peptide mixtures using a simple biuret method[J].Indian Journal of Biochemistry&Biophysics,1983,20(3):149-153.
[20]Conde E,Balboa E M,Parada M,et al.Algal proteins,peptides and amino acids[J].Functional Ingredients from Algae for Foods&Nutraceuticals,2013,61(15):135-180.
[21]Goshev I,Nedkov P.Exending the range of application of the biuret reaction:quantitative determination of insoluble proteins[J].Analytical Biochemistry,1979,95(2):340-343.
[22]Doumas B T,Bayse D D,Carter R J,et al.A candidate reference method for determination of total protein in serum.I.Development and validation[J].Clinical Chemistry,1981,27(10):1642-1650.
[23]Sapan C V,Lundblad R L,Price N C.Colorimetric protein assay techniques[J].Biotechnol Applied Biochem,1999,29(2):99-108.
[24]Lowry O H,Rosebrough N J,Farr A L,et al.Protein measurement with the Folinphenol reagent[J].Journal of Biological Chemistry,1951,193(1):265-275.
[2]李存富.关于蛋白质测定中几个问题的理论分析与讨论[J].中国酿造,2004,23(9):29-30.
[3]Friedman M.Applications of the ninhydrin reaction for analysis of amino acids,peptides,and proteins to agricultural and biomedical sciences[J].Journal of Agricultural&Food Chemistry,2004,52(3):385-406.
[4]Habeeb A F S A.Determination of free amino groups in proteins by trinitrobenzenesulfonic acid[J].Analytical Biochemistry,1966,14(3):328-336.
[5]Robbins R J,Bean S R.Development of a quantitative high-performance liquid chromatography-photodiode array detection measurement system for phenolic acids[J].Journal of Chromatography A,2004,1038(1):97-105.
[6]赵晓娟,李敏仪,黄桂颖,等.Folin-Ciocalteu法测定苹果醋饮料的总多酚含量[J].食品科学,2013,34(08):31-35.
[7]饲料添加剂酸性、中性蛋白酶活力的测定分光光度法:GB/T28715-2012[S].北京:中国标准出版社,2012.
[8]蛋白酶制剂:GB/T 23527-2009[S].北京:中国标准出版社,2009.
[9]Lubran M M.The measurement of total serum proteins by the Biuret method[J].Annals of Clinical&Laboratory Science,1978,8(2):106.
[10]Janairo G,Linley M,Yap L,et al.Determination of the Sensitivity Range of Biuret Test for Undergraduate Biochemistry Experiments[J].e-Journal of Science&Technology,2011(5):81-87.
[11]赵书景.粮食、饲料中蛋白质含量测定方法的研究[D].扬州:扬州大学,2008.
[12]Agbor G A,Vinson J A,Donnelly P E.Folin-Ciocalteau Reagent for Polyphenolic Assay[J].International Journal of Food Science Nutrition&Dietetics,2014,3(8),147-156.
[13]韩葵葵.黄酮类药物与血清蛋白的相互作用以及不同表面活性剂的影响[D].扬州:扬州大学,2008.
[14]Enechi O.C,Nwabueze Emilia C.A New Colorimetric Method for the Determination of Proteins[J].Advances in Biological Research,2013,7(5):159-162.
[15]Gornall A G,Bardawill C J,David M M.Determination of serum proteins by means of the biuret reaction[J].Journal of Biological Chemistry,1949,177(2):751-766.
[16]Daniel R M,Danson M J,Eisenthal R,et al.A new parameters controlling the effect of temperature on enzyme activity[J].Biochemical Society Transactions,2007,35(6):1536-1543.
[17]Fromm H J.The Effect of Temperature and p H on Enzyme Activity[M].Initial Rate Enzyme Kinetics.Springer Berlin Heidelberg,1975:201-235.
[18]Williams A.Mechanism of action and specificity of proteolytic enzymes[J].Quarterly Reviews Chemical Society,1969,23(1):1-17.
[19]Raghunath M,Rao B N.Determination of dipeptides in peptide mixtures using a simple biuret method[J].Indian Journal of Biochemistry&Biophysics,1983,20(3):149-153.
[20]Conde E,Balboa E M,Parada M,et al.Algal proteins,peptides and amino acids[J].Functional Ingredients from Algae for Foods&Nutraceuticals,2013,61(15):135-180.
[21]Goshev I,Nedkov P.Exending the range of application of the biuret reaction:quantitative determination of insoluble proteins[J].Analytical Biochemistry,1979,95(2):340-343.
[22]Doumas B T,Bayse D D,Carter R J,et al.A candidate reference method for determination of total protein in serum.I.Development and validation[J].Clinical Chemistry,1981,27(10):1642-1650.
[23]Sapan C V,Lundblad R L,Price N C.Colorimetric protein assay techniques[J].Biotechnol Applied Biochem,1999,29(2):99-108.
[24]Lowry O H,Rosebrough N J,Farr A L,et al.Protein measurement with the Folinphenol reagent[J].Journal of Biological Chemistry,1951,193(1):265-275.
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