Optimal conditions for assay of cytochrome-c-oxidase activity in human skeletal muscle tissue.

Abstract

Conditions for the assay of cytochrome-c-oxidase in human skeletal muscle tissue were studied, including an evaluation of the optimal conditions during the in vitro assay as well as the optimal conditions for storage and treatment of the tissue before the assay. The activity of cytochrome-c-oxidase was assayed polarographically with a Clark oxygen electrode. Optimal oxygen consumption rates were obtained at a cytochrome-c-concentration above 0.2 mmol/l, in the presence of TMPD, 2.2 mmol/l and ascorbic acid, 4.4 mmol/l as reducing agents. Enzyme proportionality was obtained after correction of the oxygen consumption rates for a blank reaction according to one of three alternatives presented. Variations in the homogenizing technique and the degree of dilution of the homogenate (1:11-1:88) had only moderate effects on the enzyme activity. Optimal storage conditions were evaluated by comparing the enzyme activities measured in fresh muscles, frozen muscles and homogenates prepared from fresh muscles. During all storage conditions significantly higher activities were obtained when sucrose buffer (0.25 mol/l) was used as homogenizing medium as compared to phosphate buffer (0.1 mol/l). Freezing and thawing of the muscle tissue before the assay caused an average decrease in the enzyme activity of 50% (P less than 0.005) as compared to the activity obtained in fresh tissue. This untoward effect of the freezing and thawing was reduced when the enzyme activity was analysed in frozen homogenates prepared from fresh muscles. Thus, an average decrease in the enzyme activity of 15% (P less than 0.05) was found in frozen homogenates prepared in sucrose buffer as compared to the activity obtained in the fresh tissue. These findings emphasize the importance of evaluating the effects of the tissue treatment for comparative studies of maximum enzyme activities in vitro.