The denaturation of haemoglobin in dilute solution

The homogenate was subjected to centrifugation at 15 OOOg,, for IOmin and the residue was then washed with buffer A and re-centrifuged. The combined supernatant fractions were centrifuged at 23000g,,. for 60min and then stirred for 20min with DEAE-cellulose ( IOOml settled volume) that had been equilibrated in buffer A. The slurry was then poured into a column (lOx4cm) and washed with buffer A (500mI). Reductase activity was then eluted with buffer A containing O.~M-KCI. Active fractions were pooled and applied at a flow rate of O.Sml/min to a column (1.4 x 8cm) of Matrex Gel Blue A (Cibacron Blue F3GA covalently linked to agarose; Amicon Corporation, High Wycombe, Bucks., U.K.). The gel was washed with buffer A containing 0.3wKCl until no more protein was present in the eluate. Enzyme activity was then eluted by application of a linear gradient of KCl (0.3 M-2.0~). Enzyme activity could also be eluted, but in substantially lower yield, by 5mwNADPH or folate. Peak enzyme activity emerged at a concentration of 1.2h1-KCl. The purified enzyme was concentrated by ultrafiltration and stored at -2OOC. Immobilized Cibacron Blue F3GA has been used in the purification of many nucleotide-dependent enzymes and may interact at the nucleotide-binding sites of these enzymes (Thompson et al., 1975). The related dye Procion Red HE3B has also been used for purification of nucleotide-dependent enzymes (Stockton et al., 1978; Whittle & Turner, 1978; Watson et al., 1978) and may exhibit preference for NADP+requiring enzymes (Watson et al., 1978). Since methylenetetrahydrofolate reductase uses NADPH as a cofactor, we have compared interactions of this enzyme with immobilized Procion Red HE3B. The enzyme could be adsorbed to Matrex Gel Red A (Procion Red HE3B-agarose) and was eluted at a similar concentration of KCI to that found for Matrex Gel Blue A. The free dyes were potent inhibitors of enzyme activity (K, < 1 ~ U M for both dyes). The enzyme could be further purified & gel filtration but lost substantial activity during this process. The molecular weight of the native enzyme was estimated to be 180000, and the enzyme retained both menadione reductase activity and tetrahydro-8-carboline synthetase activity during purification. The significance of these observations has been discussed elsewhere (Turner, 1977). Modifications of folate metabolism in brain have been implicated in a number of neurological disorders including epileptic seizures and schizophrenia (see e.g. Turner, 1977). However, anti-convulsant drugs of a wide range of structures were found not to afTect methylenetetrahydrofolate reductase activity. One must therefore look elsewhere for the site of interaction of anti-convulsant drugs with folate metabolism. In view of the reported deficiency of methylenetetrahydrofolate reductase in a schizophrenic patient (Mudd & Freeman, 1974), we have investigated the activity of the enzyme in discrete brain reaions from deceased normal and schizophrenic patients. No significant differences in activity were observed between the two groups, nor did a range of neuroleptic drugs affect the activity of the enzyme from human or ox brain. Methylenetetrahydrofolate reductase is the rate-limiting step in the biosynthesis of S-adenosylmethionine in brain (Turner, 1979) and the liver reductase has been shown to be inhibited by S-adenosylmethionine (Kutzbach & Stokstad, 1971). An inhibition constant (K,) for S-adenosylmethionine in the range 0 .4-0 .7m~ was estimated for the brain reductase, which is considerably weaker inhibition than has been reported for the liver enzyme. However, we cannot as yet exclude the possibility of desensitization of the brain enzyme to inhibition during its isolation. Since physiological concentrations of S-adenosylmethionine have been reported to be in the millimolar range in brain (Baldessarini & Kopin, 1963) our results would implicate methylenetetrahydrofolate reductase in the fine control of S-adenosylmethionine levels and hence neurotransmitter methylation in brain.