Application of a direct spectrophotometric assay employing a chromogenic substrate for tryptophanase to the determination of pyridoxal and pyridoxamine 5'-phosphates.

摘要: Abstract Improved procedures for the isolation of apotryptophanase and its use in estimation vitamin B-6 coenzymes are presented. An excess apoenzyme is allowed to react with limiting amounts pyridoxal-P. Estimation holotryptophanase thus formed by chromogenic substrate. S-o-nitrophenyl- l -cysteine, provides a sensitive (1–400 pmol) conveniently direct spectrophotometric assay For specific pyridoxamine 5′-phosphate, samples first reduced NaBH4 convert pyridoxal-P pyridoxine-P (inactive). By nonenzymatic transamination glyoxylate, pyridoxamine-P then converted quantitatively estimated apotryptophanase. The method gives excellent recoveries added indicates that many tissue extracts surpasses concentration.