Goldbeck R A, Paquette S J, Björling S C, Kliger D S
Department of Chemistry and Biochemistry, University of California at Santa Cruz 95064, USA.
Biochemistry. 1996 Jul 2;35(26):8628-39. doi: 10.1021/bi952248k.
Nanosecond absorption spectra are measured in the Soret and near-UV spectral regions of human hemoglobin (Hb) after laser photolysis of the carbonyl adduct in order to study the dynamics of globin tertiary and quaternary conformational changes. Spectra and concentrations of physical intermediates, distinguished by extent of heme ligation and intraprotein relaxation, are obtained from a global analysis using a microscopic kinetic model that explicitly accounts for six observed relaxation and recombination processes. Three observed rate constants for CO rebinding and two intraprotein relaxation constants obtained are similar to constants determined by Hofrichter et al. [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2235], the latter two comprising the 20-30-microseconds R --> T quaternary transition and a previously unassigned 1-microseconds intraprotein relaxation. On the basis of the modeled intermediate spectra, as well as UV circular dichroism results observed on this time scale [Björling, S.C., Goldbeck, R.A., Paquette, S.J., Milder, S.J., & Kliger, D.S. (1996) Biochemistry 35, 8619-8627], the 1-microsecond relaxation is assigned to heme conformational changes concomitant with a relaxation of protein conformation at the alpha 1 beta 2 interface corresponding to an initial step in a compound R --> T reaction path.
为了研究珠蛋白三级和四级构象变化的动力学，在对羰基加合物进行激光光解后，测量了人血红蛋白（Hb）在Soret和近紫外光谱区域的纳秒吸收光谱。通过使用微观动力学模型进行全局分析，获得了以血红素连接程度和蛋白质内弛豫区分的物理中间体的光谱和浓度，该模型明确考虑了六个观察到的弛豫和重组过程。观察到的三个CO重结合速率常数和两个蛋白质内弛豫常数与Hofrichter等人[(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2235]测定的常数相似，后两个常数包括20 - 30微秒的R→T四级转变和一个先前未确定的1微秒蛋白质内弛豫。基于模拟的中间体光谱以及在此时间尺度上观察到的紫外圆二色性结果[Björling, S.C., Goldbeck, R.A., Paquette, S.J., Milder, S.J., & Kliger, D.S. (1996) Biochemistry 35, 8619 - 8627]，将1微秒的弛豫归因于血红素构象变化，同时伴随着α1β2界面处蛋白质构象的弛豫，这对应于复合R→T反应路径中的初始步骤。
