A novel procedure for determining protein concentrations from absorption spectra of enzyme digests.
A novel procedure for determining protein concentrations from absorption spectra of enzyme digests.
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一种根据酶消化物的吸收光谱测定蛋白质浓度的新方法。
DOI：
10.1016/0003-2697(82)90622-4
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发表时间：
1982
影响因子：
2.9
通讯作者：
Bewley,TA
中科院分区：
文献类型：
--
作者：
Bewley,TA
已对核糖核酸酶 A、牛血清白蛋白、β-乳球蛋白 A 和 B、α-乳白蛋白、胰凝乳蛋白酶原 A 和溶菌酶测试了一种确定摩尔消光系数 (EM) 以及蛋白质浓度的新方法。 EM 值是通过结合天然蛋白质的吸收光谱和滴定至 pH 1.5 和 pH ≥ 13 的蛋白质嗜热菌蛋白酶消化物的两等分试样之间产生的差异光谱而获得的。酶消化显示出可以最大限度地减少差异光谱的构象贡献。然后根据消化物的酪氨酸摩尔浓度和先前测定的蛋白质酪氨酸含量计算天然蛋白质的 EM。这些 EM 值显示的最大误差为 -2.2%，平均绝对误差为 1.4%。小至 100 μg 的样品即可给出令人满意的结果。不需要准确的样品重量以及关于水分和灰分含量的假设。此外，还描述了使用二阶导数（二阶）吸收光谱来获取酶消化产生的光谱归一化程度。
A novel procedure for determining molar extinction coefficients (EM), and hence protein concentrations, has been tested on ribonuclease A, bovine serum albumin, β-lactoglobulins A and B, α-lactalbumin, chymotrypsinogen A, and lysozyme. EMvalues were obtained from a combination of the absorption spectrum of the native protein and a difference spectrum generated between two aliquots of a thermolysin digest of the protein titrated to pH 1.5 and pH ≥ 13. Enzymatic digestion was shown to minimize conformational contributions to the difference spectrum. The EMof the native protein is then calculated from the tyrosine molarity of the digest and the previously determined tyrosine content of the protein. These EMvalues displayed a maximum error of −2.2% and an average absolute error of 1.4%. Samples as small as 100 μg give satisfactory results. Accurate sample weight and assumptions with regard to moisture and ash content are not required. In addition, the use of second-derivative (second-order) absorption spectroscopy to access the degree of spectral normalization produced by enzymatic digestion is described.