32] Assay of triose phosphates

Abstract

Publisher Summary This chapter discusses the assay of triose phosphates. The properties of two triose phosphates—D-glyceraldehyde-3-phosphate (G-3-P) and dihydroxyacetone phosphate (DHA-P)—can be used with varying success in assaying the triose phosphates and the enzymes with which they interact. This chapter presents procedure for estimation of total triose phosphate, estimation of individual triose phosphates in mixtures, and separation of triose phosphates. The procedures most frequently used are based on (1) the determination of total alkali-labile phosphorus, (2) the assay of the methyl glyoxal formed by heating triose phosphates in acid by the colorimetric method of Ariyama, or by the procedure of Dounce and Beyer in which methyl glyoxal is further converted to acetaldehyde which is then assayed by a modification of the lactic acid method of Barker and Summerson, (3) optical changes in systems containing excess triosephosphate isomerase and either α-glycerophosphate dehydrogenase and DPNH (in the absence of phosphate or arsenate) or triosephosphate dehydrogenase, DPN, and arsenate, and (4) the spectrophotometric measurement of colored 2,4-dinitrophenylhydrazine derivatives. The 2,4-dinitrophenylhydrazine derivatives have also been measured gravimetrically, although the procedure is rather cumbersome.