Electrode measurements of the net charge on muscle proteins

Bryson, Elzbieta Anna (1997). Electrode measurements of the net charge on muscle proteins. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f5ed

Abstract

Electrode techniques for measuring Donnan potentials in protein solutions were studied and applied to elucidate the effect of methylation on the net charge of heavy meromyosin (HMM) and the effect of Ca²⁺ on the net charge of the thin filament proteins.

Drifts in potentials, observed for macroelectrodes, were examined and their cause established to be KC1 leakage out of electrodes. The microelectrode technique was applied to protein solutions and microelectrodes with resistance less than 1 MΩ were used: the conditions for manufacturing and maintaining electrodes functional were established.

HMM was isolated (from rabbit muscle) and methylated; the modification was verified by amino acid analysis. ATPase activity of methylated HMM was found to be significantly elevated in the presence of Ca²⁺ and decreased in the presence of EDTA with respect to the activity of the native protein. Values of the net charge of both proteins were determined at pH 6.7 and no significant difference was found between them. Calculations of the theoretical charge of lysine and N^ͼ-dimethyllysine were performed which indicated only 0.5% difference at pH 6.7.

F-actin, tropomyosin-troponin (Tm-tn) and reconstituted thin filaments (RTFs) were isolated from rabbit muscle. Conditions for preserving the binding between F-actin and Tm-tn were established. Net charges of F-actin, Tm-tn, RTFs and BSA (control) were measured in solutions of pCa 3.2-8.7 at ionic strength 0.02 M and pH 7.0. Significant decrease in the negative charge of the RTFs, Tm-tn and F-actin was observed with increasing concentrations of free Ca²⁺, between pCa 6.5 and 3 approximately.

Values of the molecular and specific charge at pH 7.0 and the isoelectric point were calculated from the amino acid sequences of the main muscle proteins.