Effect of pH on the adsorption and activity of creatine phosphokinase.
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.
Pancera
Sabrina M
SM
Instituto de Química, Universidade de São Paulo, P.O. Box 26077, São Paulo, SP, 05513-970, Brazil.
Gliemann
Hartmut
H
Schimmel
Thomas
T
Petri
Denise F S
DF
D000327:Adsorption; D003402:Creatine Kinase / Q000737:chemistry*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force / Q000379:methods; D010316:Particle Size; D012680:Sensitivity and Specificity; D012825:Silicon / Q000737:chemistry; D013499:Surface Properties; D013997:Time Factors
D003402:Creatine Kinase; D012825:Silicon