Poli Alessandra Lima, Moreira Leonardo Marmo, Hidalgo Angel Alberto, Imasato Hidetake
Instituto de Química de São Carlos, Universidade de São Paulo, Brazil.
Biophys Chem. 2005 Apr 22;114(2-3):253-60. doi: 10.1016/j.bpc.2004.12.041. Epub 2005 Jan 15.
The complex oligomeric assembly of the hemoglobin subunits may influence the autoxidation rate. To understand this relation, the rate of autoxidation was studied at pH 9.0, where the Glossoscolex paulistus Hemoglobin (GpHb) dissociates. At alkaline pH, this hemoglobin is dissociated into monomers, trimers and tetramers, allowing the study of the integral protein and monomer subunit autoxidation on independent experiments. The autoxidation rate was evaluated in the presence and absence of cyanide (CN(-)), a strong field ligand to the ferric ion. The oxidation kinetic was monitored using the UV-vis absorption at 415 nm, and resulted in: i) bi-exponential kinetics for the whole hemoglobin (indicating a fast and a slow oxidative process) and ii) mono-exponential for the monomer (indicating a single process). To understand the specific characteristics of each autoxidation process, Arrhenius plots allowed the determination of the activation energy. The experimental results indicate for the whole hemoglobin in the absence of CN(-) an activation energy of 150 +/- 10 kJ mol(-1) for the fast and the slow processes. Under the same conditions the monomer displayed an activation energy of 160 +/- 10 kJ mol(-1), very close to the value obtained for the integral protein. The pseudo-second order rate constant for the whole protein autoxidation by CN(-) showed two different behaviors characterized by a rate constant k(CN1)' = 0.11 +/- 0.02 s(-1) mol(-1) L for CN(-) concentrations lower than 0.012 mol L(-1); and k(CN1)" = 0.76 +/- 0.04 s(-1) mol(-1) L at higher concentrations for the fast process, while the slow process remain constant with k(CN2) = 0.033 +/- 0.002 s(-1) mol(-1) L. The monomer has a characteristic rate constant of 0.041 +/- 0.002 s(-1) mol(-1) L for all cyanide concentrations. Comparing the results for the slow process of the whole hemoglobin and the oxidation of the monomer, it is possible to infer that the slow process has a strong contribution of the monomer in the whole hemoglobin kinetic. Moreover, as disulfide linkers sustain the trimer assembly, cooperativity may explain the higher kinetic constant for this subunit.
血红蛋白亚基的复杂寡聚体组装可能会影响自氧化速率。为了理解这种关系，我们在pH 9.0的条件下研究了自氧化速率，此时圣保罗巨舌蛭血红蛋白（GpHb）会发生解离。在碱性pH条件下，这种血红蛋白会解离成单体、三聚体和四聚体，从而可以在独立实验中研究完整蛋白质和单体亚基的自氧化。在存在和不存在氰化物（CN(-)）的情况下评估自氧化速率，氰化物是铁离子的强场配体。使用415 nm处的紫外可见吸收监测氧化动力学，结果如下：i）完整血红蛋白呈现双指数动力学（表明存在快速和慢速氧化过程），ii）单体呈现单指数动力学（表明只有一个过程）。为了理解每个自氧化过程的具体特征，通过阿累尼乌斯图确定了活化能。实验结果表明，在不存在CN(-)的情况下，完整血红蛋白快速和慢速过程的活化能为150±10 kJ mol(-1)。在相同条件下，单体的活化能为160±10 kJ mol(-1)，与完整蛋白质获得的值非常接近。CN(-)对完整蛋白质自氧化的伪二级速率常数表现出两种不同行为，对于低于0.012 mol L(-1)的CN(-)浓度，快速过程的速率常数k(CN1)' = 0.11±0.02 s(-1) mol(-1) L；对于较高浓度，快速过程的速率常数k(CN1)" = 0.76±0.04 s(-1) mol(-1) L，而慢速过程保持恒定，k(CN2) = 0.033±0.002 s(-) mol(-1) L。对于所有氰化物浓度，单体的特征速率常数为0.041±0.002 s(-1) mol(-1) L。比较完整血红蛋白慢速过程和单体氧化的结果，可以推断出慢速过程在完整血红蛋白动力学中单体的贡献很大。此外，由于二硫键连接维持三聚体组装，协同作用可能解释了该亚基较高的动力学常数。
