Adsorption Dynamics of a Phospholipase A2 onto a Mercury Electrode Surface

Abstract

Rapid cyclic voltammetry has been employed to investigate the electrochemical behavior of porcine pancreatic phospholipase A2 (PLA2) adsorbed onto a hanging mercury drop electrode surface. The electrochemical response of PLA2 exhibits a pair of voltammetric peaks which are ascribed to the reductiodoxidation of cystinekysteine residues within the enzyme strongly adsorbed on mercury surfaces over the pH range of 0.77-8.50. Based on theoretical fits to the data, the adsorption process, under all experimental conditions, is found to be kinetically controlled. In addition, the adsorption appears to be consistent with a Frumkin adsorption isotherm with a small repulsive interaction. Both pH and temperature have a dramatic effect on the adsorption behavior in terms of the adsorption rate, equilibrium coverage, and molecular conformation. Introductionof the effects of sweep rate, concentration, pH, and temperature,

Reactions of Phospholipase A2 at a Mercury Electrode Surface

“…In contrast to the experimental value of 71 mV, 7 this reaction would be expected to exhibit a 59 mV shift of the formal potential (at 25 °C) per pH unit change as predicted by the Nernst equation. As to the origin of this difference, it was difficult to ascertain without a better understanding of the reaction mechanism (more detailed discussion below).…”

“…The experimental apparatus and data analysis for dc cyclic voltammetry have been described previously. 7 In the ac voltammetry experiments, the sine wave was generated with a Tektronix CFG 250 function generator, with a typical frequency of 73 Hz and a peak-to-peak amplitude of 10 mV. This ac signal was superimposed onto a 10 mV/s dc ramp which was generated with an EG&G PARC Model 175 universal programmer.…”

“…Table 3 summarizes the results for C 1 and C f at various solution pH values, where it can be seen that C 1 is about 6 µF/cm 2 , corresponding to an effective dielectric constant of about 6 by taking into account the adsorbed molecular dimensions. 7,12 One can also observe that, on the more negative side of the pseudocapacitance peak, there is another ac current minimum which corresponds to the reduced state of PLA 2 , i.e., RSH, and this current is approximately equal to that at E 2 ) -0.52 V, indicating that the overall interfacial dielectric properties of the adsorbed PLA 2 molecules were practically unchanged before and after the redox reaction. Although inferring structural information from capacitance measurements must be done with great care, what one can say is that based on these capacitance results, there appear to be no gross changes in the interfacial structure of adsorbed PLA 2 which is consistent with our previous suggestion that its overall molecular conformation is preserved during the adsorption and electron transfer processes.…”

“…We recently carried out some studies on the adsorption dynamics of porcine pancreatic phospholipase A 2 (PLA 2 ) onto a hanging mercury drop electrode (HMDE) surface. 7 Since this study suggested that the adsorption was through a cystine residue within the enzyme, we wanted to probe the surface reactions involved, thereby providing additional insight into the current understanding of the thiol/disulfide chemisorption. We have carried out additional studies of this system with both dc and ac cyclic voltammetric techniques, in terms of the surface reaction mechanism, electron transfer kinetics, and the electrical properties of the adsorbed PLA 2 molecules.…”

Enzymatic Activity of a Phospholipase A2: An Electrochemical Approach

“…Recently, new methodologies have been developed which allow ready fabrication of monolayer films of phospholipids onto electrode surfaces, and these have paved the way for the study of the enzymatic activity of PLA 2 onto solid-supported phospholipid substrates. In an effort to develop a novel approach for evaluating the PLA 2 enzymatic activity with electrochemical techniques, we have previously carried out studies on the adsorption dynamics and the surface reactions of porcine pancreatic phospholipase A 2 onto a mercury electrode surface. , Such preliminary studies are essential for a more thorough understanding of the behavior of the phospholipases A 2 at the electrode/solution interface. It has been previously demonstrated that PLA 2 adsorbs strongly onto mercury electrode surfaces, and it was speculated that the adsorption site is one of the seven disulfide groups, likely the Cys61−Cys91, which is very close to the N-terminus of the enzyme molecule and the interfacial recognition site. ,,, …”