Laurenti E, Suriano G, Ghibaudi E M, Ferrari R P
Dipartimento di Chimica I.F.M., Università di Torino, Italy.
J Inorg Biochem. 2000 Oct 1;81(4):259-66. doi: 10.1016/s0162-0134(00)00124-0.
The effects of chloride, dihydrogenphosphate and ionic strength on the spectroscopic properties of horseradish peroxidase in aqueous solution at pH=3.0 were investigated. A red-shift (lambda=408 nm) of the Soret band was observed in the presence of 40 mM chloride; 500 mM dihydrogenphosphate or chloride brought about a blue shift of the same band (lambda=370 nm). The EPR spectrum of the native enzyme at pH 3.0 was characterized by the presence of two additional absorption bands in the region around g=6, with respect to pH 6.5. Chloride addition resulted in the loss of these features and in a lower rhombicity of the signal. A unique EPR band at g=6.0 was obtained as a result of the interaction between HRP and dihydrogenphosphate, both in the absence and presence of 40 mM Cl-. We suggest that a synergistic effect of low pH, Cl- and ionic strength is responsible for dramatic modifications of the enzyme conformation consistent with the Fe(II)-His170 bond cleavage. Dihydrogenphosphate as well as high chloride concentrations are shown to display an unspecific effect, related to ionic strength. A mechanistic explanation for the acid transition of HRP, previously observed by Smulevich et al. [Biochemistry 36 (1997) 640] and interpreted as a pure pH effect, is proposed.
研究了氯离子、磷酸二氢根和离子强度对pH = 3.0水溶液中辣根过氧化物酶光谱性质的影响。在存在40 mM氯离子的情况下，观察到Soret带发生红移（λ = 408 nm）；500 mM磷酸二氢根或氯离子会导致同一谱带发生蓝移（λ = 370 nm）。相对于pH 6.5，pH 3.0时天然酶的电子顺磁共振（EPR）谱的特征是在g = 6附近区域存在两个额外的吸收带。添加氯离子导致这些特征消失，信号的菱形度降低。在不存在和存在40 mM Cl-的情况下，由于辣根过氧化物酶与磷酸二氢根之间的相互作用，在g = 6.0处获得了一个独特的EPR谱带。我们认为，低pH、Cl-和离子强度的协同作用导致了与Fe(II)-His170键断裂一致的酶构象的显著改变。磷酸二氢根以及高氯离子浓度显示出与离子强度相关的非特异性效应。提出了对辣根过氧化物酶酸转变的机理解释，此前Smulevich等人[《生物化学》36 (1997) 640]观察到该转变并将其解释为纯粹的pH效应。
