埃氏消化链球菌脱辅基黄素氧还蛋白与黄素单核苷酸结合的研究：pH值和氯化钠浓度依赖性

Studies on the binding of FMN by apoflavodoxin from Peptostreptococcus elsdenii, pH and NaCl concentration dependence.

Gast R, Valk B E, Müller F, Mayhew S G, Veeger C

Biochim Biophys Acta. 1976 Oct 28;446(2):463-71. doi: 10.1016/0005-2795(76)90012-x.

Abstract

The pH and ionic strength dependence of the interaction of FMN with apoflavodoxin has been studied by fluorometry in the pH region 2-5, at 22 degrees C. 2. The rate constant of dissociation and the dissociation constant were experimentally determined; the rate constants of association were claculated at a given pH value. These constants depend on the ionic strength. The plots of these constants against the square root of the ionic strength are straight. 3. Our data have been interpreted in terms of the Brönsted theory, which relates chemical reaction rates to ionic strength. The data indicate that the apoenzyme reaches its maximum net positive charge at pH 2.0-2.6. The calculated net charge in this pH region is between 11 and 12 and is in agreement with the theoretical value of 12 as deduced from the primary structure of the protein. The isoelectric point of the holoenzyme is about 4. 4. The rate constant of association extrapolated to zero ionic strength is 3.2-10(5)M-1-s-1 and is pH-independent. 5. The rate constant of dissociation and the dissociation constant extrapolated to zero ionic strength depend on the pH. The results are explained by assuming that there are two protein ionizations with a pK value of 3.4; these ionizing groups are possibly close to the FMN binding site.

摘要

采用荧光分析法在22℃、pH值为2至5的范围内研究了黄素单核苷酸（FMN）与脱辅基黄素odoxin相互作用对pH值和离子强度的依赖性。2. 通过实验测定了解离速率常数和解离常数；在给定的pH值下计算缔合速率常数。这些常数取决于离子强度。这些常数对离子强度平方根的作图呈直线关系。3. 我们的数据已根据布朗斯特理论进行了解释，该理论将化学反应速率与离子强度联系起来。数据表明，脱辅基酶在pH值为2.0至2.6时达到其最大净正电荷。在该pH范围内计算出的净电荷在11至12之间，与根据蛋白质一级结构推导的理论值12一致。全酶的等电点约为4。4. 外推至零离子强度时的缔合速率常数为3.2×10⁵ M⁻¹·s⁻¹，且与pH值无关。5. 外推至零离子强度时的解离速率常数和解离常数取决于pH值。通过假设存在两个pK值为3.4的蛋白质电离来解释这些结果；这些电离基团可能靠近FMN结合位点。