Pancera Sabrina M, Gliemann Hartmut, Schimmel Thomas, Petri Denise F S
Instituto de Química, Universidade de São Paulo, P.O. Box 26077, São Paulo, SP, 05513-970, Brazil.
J Phys Chem B. 2006 Feb 16;110(6):2674-80. doi: 10.1021/jp0532364.
The combination of in situ ellipsometry with atomic force microscopy in the liquid for the study of adsorption of creatine phosphokinase (CPK) onto silicon wafers was shown for the first time. The thickness, adsorbed amount, and topographic information of the adsorbed CPK layers were obtained under different pH conditions. The thickness values of adsorbed CPK layer determined by both techniques were in excellent agreement. At pH 4, CPK monomers present in solution adsorb, forming a very thin (approximately 0.8 nm) layer, indicating CPK unfolding. Upon increasing the pH to 6.8, the adsorbed layer is composed of a mixture of CPK dimers (native structure) and intermediates, increasing the film thickness (approximately 2.4 nm). At pH 9, CPK dimers form monolayers with the highest thickness (approximately 4.0 nm). The nature of interactions between CPK and Si wafers associated with the hydration force seems to control the degree of CPK unfolding upon adsorbing. The enzymatic activity of free CPK and of adsorbed CPK at pH 4, pH 6.8, and pH 9 was measured as a function of pH. In comparison to free CPK in solution, adsorbed CPK presented a shift of the optimal pH from 6.8 toward alkaline pH.
首次展示了将原位椭偏仪与液体中的原子力显微镜相结合用于研究肌酸磷酸激酶（CPK）在硅片上的吸附情况。在不同pH条件下获得了吸附的CPK层的厚度、吸附量和形貌信息。两种技术测定的吸附CPK层的厚度值非常吻合。在pH 4时，溶液中存在的CPK单体吸附，形成非常薄（约0.8 nm）的层，表明CPK发生了去折叠。将pH提高到6.8时，吸附层由CPK二聚体（天然结构）和中间体的混合物组成，膜厚度增加（约2.4 nm）。在pH 9时，CPK二聚体形成厚度最高（约4.0 nm）的单层。与水合力相关的CPK与硅片之间相互作用的性质似乎控制了吸附时CPK的去折叠程度。测定了游离CPK和在pH 4、pH 6.8和pH 9下吸附的CPK的酶活性随pH的变化。与溶液中的游离CPK相比，吸附的CPK的最佳pH从6.8向碱性pH偏移。
