Studies on Cenditions for Measurement of Human LD Activity

Abstract

Optimum reaction conditions at 37° for spectrophotometric assay of activity (from lactate to pyruvate) of human LDH isoenzymes were determined with respect to buffer concentration, NAD concentration, substrate concentration and pH.
Human LDH isoenzymes used were separated by column chromatography.
Optimum concentration of Glycine-NaOH buffer were within the range of 0.1M to 0.2M for LDH₁ and LDH₅ isoenzymes. An optimum activity was obtained at a NAD concentration of 4.5×10^-3M. optimum concentration of DL-lactate was 0.25M for total LDH, since it was 0.1M for LDH₁and0.5M for LDH₅.
Activities of LDH₁and LDH₅were enhanced with the increase of pH, when the activities were compared between of 9.0-10.8.
Although Fendley reported that the LDH5 isoenzyme was unable to measure when the assay was performed of pH 10.0, we have found that the LDH₅ isoenzyme activity can measure when a LDH·NAD complex is formed by a preincubation for 5min. and the reaction is started by a substrate buffer.