Barker P D, Mauk M R, Mauk A G
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Biochemistry. 1991 Mar 5;30(9):2377-83. doi: 10.1021/bi00223a012.
The proton titration curves of yeast iso-1-ferricytochrome c and selected point mutants of this protein have been determined between pH 3 and 11 at 10 and 25 degrees C with a computer-controlled titration system. Initial titration of the wild-type protein to acidic pH followed by subsequent titrations to alkaline and then acidic pH demonstrates hysteresis, with one more group (28.7) titrating between pH 11 and 3 than originally titrated (27.7) between pH 3 and 11. Initial titration to alkaline pH, however, resulted in observation of the same number of groups in both directions of titration (28.7 vs 28.6). At 10 degrees C, 7.5 fewer groups were found to titrate over the same range of pH. Titration curves obtained for six cytochrome c mutants modified at Arg-38, Phe-82, Tyr-48, and Tyr-67 were analyzed by subtraction of the corresponding titration curve for the wild-type protein to produce difference titration curves. In most cases, the effects of these mutations as revealed in the difference titration curves could be accounted for as either the result of introduction of an additional group titrating within this pH range, the result of a change in the pK of a titrating residue, and/or the result of a change in the pK for either the first acidic or the first alkaline protein conformational transition. In addition to demonstration of the electrostatic consequences of the mutations in cytochrome c studied here, this study establishes the general usefulness of precise proton titration curve analysis in the characterization of variant proteins produced through recombinant genetic techniques.
利用计算机控制的滴定系统，在10℃和25℃下测定了酵母同工酶-1-铁细胞色素c及其该蛋白的选定定点突变体在pH 3至11之间的质子滴定曲线。将野生型蛋白初始滴定至酸性pH，随后滴定至碱性pH，再滴定至酸性pH，结果显示出滞后现象，在pH 11至3之间滴定的基团比最初在pH 3至11之间滴定的基团多一个（28.7个）。然而，初始滴定至碱性pH时，在两个滴定方向上观察到的基团数量相同（28.7对28.6）。在10℃时，发现在相同pH范围内滴定的基团少7.5个。通过减去野生型蛋白相应的滴定曲线来分析在Arg-38、Phe-82、Tyr-48和Tyr-67处修饰的六个细胞色素c突变体获得的滴定曲线，以产生差异滴定曲线。在大多数情况下，差异滴定曲线中显示的这些突变的影响可以解释为是在此pH范围内引入了一个额外的滴定基团、滴定残基的pK发生变化以及/或者第一个酸性或第一个碱性蛋白构象转变的pK发生变化的结果。除了证明此处研究的细胞色素c突变的静电后果外，本研究还确立了精确质子滴定曲线分析在表征通过重组遗传技术产生的变体蛋白方面的普遍实用性。
