Straume M, Mitchell D C, Miller J L, Litman B J
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
Biochemistry. 1990 Oct 2;29(39):9135-42. doi: 10.1021/bi00491a006.
Flash photolysis experiments designed to monitor the establishment of the metarhodopsin I to metarhodopsin II equilibrium are interpreted according to a branched model in which two spectrally indistinguishable but kinetically distinguishable forms of metarhodopsin II are postulated to exist in equilibrium with a common pool of metarhodopsin I. This interpretation arises from the consistent requirement for at least three exponentials for a valid description of the observed growth of absorbance at 380 nm following bleaching of bovine rhodopsin in rod outer segment disk membranes. Analysis of the 380-nm transient absorbance data permitted direct determination of the five physically interpretable individual rate constants of the model. This analysis represents a more explicit interpretation of kinetic data than that employed in earlier experiments of this kind, which involved estimating only apparent rates and apparent amplitudes of discrete multiexponential functions. The 380-nm absorbance contributions of all relevant species contributing to the observed dynamic absorbance change were accounted for simultaneously during nonlinear least-squares estimation of the model rate parameters. Analysis of deconvoluted equilibrium spectra acquired from samples identical with those used in the kinetics experiments confirmed the metarhodopsin I-metarhodopsin II equilibrium constants, Keq, derived from the dynamic analyses. It is shown that Keq varies from 1.28 at 10 degrees C to 7.3 at 37 degrees C and that approximately 90% of the metarhodopsin II present is in the form of metarhodopsin IIslow over the temperature range 10-37 degrees C. A physical interpretation of this decay model is discussed in the context of a distribution of metarhodopsin II structural and energetic states.
旨在监测视紫红质I到视紫红质II平衡建立的闪光光解实验，是根据一个分支模型来解释的。在该模型中，假定存在两种光谱上无法区分但动力学上可区分的视紫红质II形式，它们与视紫红质I的一个共同池处于平衡状态。这种解释源于对至少三个指数的一致要求，以便有效描述在杆状外段盘膜中牛视紫红质漂白后，在380nm处观察到的吸光度增长。对380nm瞬态吸光度数据的分析允许直接确定该模型的五个可从物理角度解释的个体速率常数。这种分析比早期此类实验中采用的方法更明确地解释了动力学数据，早期实验仅涉及估计离散多指数函数的表观速率和表观幅度。在对模型速率参数进行非线性最小二乘估计期间，同时考虑了所有对观察到的动态吸光度变化有贡献的相关物种在380nm处的吸光度贡献。对从与动力学实验中使用的样品相同的样本获得的去卷积平衡光谱的分析，证实了从动态分析得出的视紫红质I - 视紫红质II平衡常数Keq。结果表明，Keq在10℃时为1.28，在37℃时为7.3，并且在10 - 37℃的温度范围内，大约90%的视紫红质II以视紫红质II慢形式存在。在视紫红质II结构和能量状态分布的背景下，讨论了这种衰变模型的物理解释。
