消光系数法与二喹啉甲酸法测定多肽浓度精密度的比较

Comparative Study on Precision of the Extinction Coefficient Method and the Bicinchoninic Acid Methodin Determination of Polypeptide Concentration

目的比较研究实验室测定多肽浓度的消光系数法与二喹啉甲酸法(bicinchoninic acid,BCA)的精密度。方法将牛血清白蛋白(bovine serum albumin,BSA)与固相合成环肽CYE(NH2-CYENLRSTC-COOH)、CLP(NH2-CLPLWYPSC-COOH)及CHY(NH2-CHYPTYSKC-COOH)溶液梯度稀释后分别使用消光系数法和BCA法进行吸光度测定,通过消光系数公式或标准曲线方程式计算相应浓度值,比较浓度计算值与理论值之间的差异,并计算测定方法的精密度。结果消光系数法的浓度计算值与理论值之间差异无统计学意义(P>0.05)。BCA法中,以BSA作为标准品得到的多肽浓度计算值与理论值之间有显著差异(P<0.05);以待测多肽自身为标准品求得的多肽浓度计算值与理论值之间无明显差异(P>0.05);以与待测多肽长度相同、分子量相近的多肽绘制个性化标准曲线求得的多肽浓度计算值与理论值之间存在统计学差异(P<0.05),但求得的浓度计算值相比BSA标准曲线的浓度计算值更接近理论值(P<0.05)。各方法的精密度之间差异无统计学意义(P>0.05)。结论消光系数法推荐用于含有色氨酸和酪氨酸的多肽;当标准品在结构、疏水性等方面的性质越接近待测物,使用个性化标准曲线的BCA法得到的浓度计算值越准确,可用于多肽浓度的快速半定量测定。

Objective To compare the precision of the extinction cofficient method and the bicinchoninic acid(BCA)method in determination of polypeptide concentration.Methods The solutions of bovine serum albumin(BSA)and the cyclic peptides of CYE(NH2-CYENLRSTC-COOH),CLP(NH2 CLPLW YPSC-COOH)and CHY(NH2-CHYPTYSKC-COOH)were synthesized using solid phase method,the solutions were then diluted in gradient.The extinction coefficient method and the BCA method were used to measure the absorbance of the solutions.The concentration was calculated by the extinction coefficient formula or the standard curve equation.The differences between the calculated value and the theoretical value were compared.The precisions of the measurement methods were compared.Results There was no significant difference in concentrations between the calculated value obtained by extinction coefficient method and the theoretical value(P>0.05).In the BCA method,there was a significant difference between the theoretical value and the calculated value when BSA was used as a standard(P<0.05).There was no significant difference between the calculated value and theoretical value when the polypeptide itself was used as a standard(P>0.05).There was a significant difference between the calculated value and theoretical value according to the individual-ized standard curve plotted through polypeptides which had an approximate length and molecular weight compared to the polypeptide to be tested(P<0.05).However,this calculated value was closer to the theoretical value than that calculated through BSA standard curve(P<0.05).There was no significant difference in precision between the two methods(P>0.05).Conclusion The extinction coefficient method is suggested when the polypeptide contains tryptophan or tyrosine.When the standard substance is closer to the substance to be measured in terms of structure and hydrophobicity,the concentration calculated by BCA method using individualized standard curve is more accurate,which can be used for rapid semi-quantitative determination of polypeptide concentration.