Molecular titration as a means of calibrating enzyme reference materials

Abstract

Active site titration provides a means of calibrating enzyme reference materials in molecular concentration units independent from the incubation conditions used in kinetic assavs. Such reference materials may serve as primary standards for calibrating any kinetic assay using the same active site. Active site titration of aspartate aminotransferase has been done by fluorimetric measurement of the half-cycle transamination of the phosphopyridoxal form. Another promising approach is the stoichiometric titration with specific suicide substrates such as vinylglycine. Expression of results in molecular concentration units requires that both the primary enzyme standard and the enzyme as measured in blood plasma show similar turnover numbers and substrate specificity in the kinetic assay being used. This is best achieved with purified reference materials of human origin. If the assay in plasma measures the sum of several isoenzymes having different turnover numbers, then the calibration is no longer absolute but becomes method-dependent.