White H D
Department of Biochemistry, University of Arizona, Tucson 85721.
Biochemistry. 1988 May 3;27(9):3357-65. doi: 10.1021/bi00409a036.
Calcium binding to whiting parvalbumin induces large changes in the fluorescence, absorption, and circular dichroism spectra of the protein. The fluorescence emission maximum of the single tryptophan shifts from 325 to 348 nm upon the removal of calcium and decreases in intensity by 50%. All of the spectral changes are linear between 0 and 2 mol of calcium bound/mol of protein, which suggests that the only protein species present in significant concentration are PA0 and Pa-Ca2. The kinetics of calcium binding measured by stopped-flow fluorescence are accurately single exponential from 2 X 10(-7) to 2 X 10(-4) M free calcium. The kinetics of calcium dissociation show a pronounced lag and are best fit by two rate constants of 1.2 and 3.0 s-1. The minimal kinetic mechanism that adequately describes the rate and equilibrium data is a branched pathway mechanism in which the rate and equilibrium constants are markedly different for each pathway: (formula; see text) At [Ca] less than 2 microM the upper kinetic pathway of calcium binding predominates whereas at [Ca] greater than 2 microM calcium binding occurs predominantly by the lower kinetic pathway. Calcium dissociates primarily by the upper kinetic pathway.
钙与牙鳕小清蛋白结合会引起该蛋白质荧光、吸收和圆二色光谱的显著变化。去除钙后，单一色氨酸的荧光发射最大值从325纳米移至348纳米，强度降低50%。在每摩尔蛋白质结合0至2摩尔钙之间，所有光谱变化都是线性的，这表明大量存在的唯一蛋白质种类是PA0和Pa-Ca2。通过停流荧光法测得的钙结合动力学在游离钙浓度为2×10⁻⁷至2×10⁻⁴ M时准确呈单指数形式。钙解离动力学表现出明显的滞后现象，最适合用1.2和3.0 s⁻¹的两个速率常数来拟合。能充分描述速率和平衡数据的最小动力学机制是一种分支途径机制，其中每个途径的速率和平衡常数明显不同：（公式；见正文）在[Ca]小于2微摩尔时，钙结合的上动力学途径占主导，而在[Ca]大于2微摩尔时，钙结合主要通过下动力学途径发生。钙主要通过上动力学途径解离。
