Determination of serine hydroxymethyltransferase and reduced folate pools in tissue extracts

Serine hydroxymethyltransferase (SHMT) from all sources tested catalyzes the slow exchange of the pro-2S proton of glycine with solvent protons. In the presence of tetrahydrofolate (H(4)PteGlu(n)) this exchange rate is increased by about three orders of magnitude. This H(4)PteGlu(n)-dependent exchange has been developed into a rapid and sensitive assay for both SHMT and H(4)PteGlu(n) and the one-carbon derivatives of H(4)PteGlu(n). The procedure involves incubating [2-H-3]glycine, H(4)PteGlu(n), and SHMT for 3 min followed by a separation of the exchanged protons in the solvent from the substrate glycine on a small Dowex-50 cation-exchange column at pH 2. In the presence of an excess of H(4)PteGlu(n) the exchange rate is proportional to nanogram levels of SHMT. In the presence of an excess of SHMT the exchange rate is directly proportional to the concentration of H(4)PteGlu(n) in the 0.1 to 1 pmol range. The concentration of one-carbon derivatives of H(4)PteGlu(n) is determined by a preincubation of cell extracts with enzymes that convert each derivative into H(4)PteGlu(n). A complete reduced folate pool analysis of a tissue extract can be obtained in less than 2 h once a standard curve has been prepared for H(4)PteGlu(n). The method does not distinguish between mono- and polyglutamate forms of the coenzyme. (C) 1997 Academic Press.