纯化HuIFN-α蛋白含量测定方法的研究

Studies on determination method of the purified HuIFN-α protein

本文对Kjeldah1氏 ,Lowry氏及Kalckar氏三种测定纯化α型人白细胞干扰素 (HuIFN α)蛋白质含量的方法进行了比较试验和研究。Kjeldah1氏法测定结果准确可靠 ,但样品用量大 ,费时 ;Lowry氏法与Kjeldah1结果一致 ,样品用量少 ,结果稳定 ;Kalckar氏法操作简单方便 ,样品用量少 ,但测定误差较大 ,作者改良了Kalckar氏法 ,测定误差大为下降。纯化HuIFN α与人血清白蛋白 (HSA) ,牛血清白蛋白 (BSA)一样在 2 78nm波长处有特征吸收峰 ;它们的Folin酚试剂显色液最大吸收波长均在 75 8nm附近 ;Lowry氏法的显色温度对显色强度及标准曲线的斜率有较大影响 ;当纯化HuIFN α中残余硫氰酸根 (CNS)含量超过 10 0 μg/ml时 ,对Lowry氏法的测定有明显干扰。

Exploring the method of determination of the purified HuIFN α protein in the present paper,the author does an experiment of comparing Kjeldah1 method,Lowry method and Kalckar method which are used to determinate the concentration of the purified HuIFN α protein and makes a further study.Both Kjeldh1 method and Lowry method have the some allurate results but the latter needs less samples and time.Kalckar method simply and needs less Samples but has larger determination errors which has been greatly reduced by author using the reformatory Kalckar method.The purified HuIFN α protein has the same characteristic absorption peak as the HSA and the BSA at wavelengths 278 nm;their developing dye solution of Folin reagent all have maximum absorption wavelengths about 758 nm.Lowy methods chromogenic temperature greatly effects on its chromogenic intension and the slope of standard curve;CNS which remains in purified HuIFN α protein will interere in the determination of Lowry method when its concentration is beyond 100 μg/ml