pH-Stat titration method for dihydrofolate reductase activity measurements: application to determination of substrate Michaelis constant and antifolate inhibition constant

Abstract

A pH-Stat titration method was developed for measuring dihydrofolate reductase (DHFR) activity; this method permits detection of very low DHFR activities corresponding to 100 pmol of substrate reduced per minute. This value is about ten times lower than those observed using the classical spectrophotometric method. This sensitivity makes it possible to measure the DHFR in crude tissue extracts. With beef liver DHFR, Michaelis constants for the cofactor NADPH and the natural substrate determined by this method were 1.9 +/- 0.3 X 10(-5) and 8.5 +/- 0.5 X 10(-7) M, respectively. The inhibition constant of methotrexate, a competitive inhibitor of dihydrofolate, was 3.4 +/- 1.3 X 10(-11) M.