An Improved Colorimetric Assay for Pyridoxal Phosphate Using Highly Purified Apotryptophanase
Methods for the analysis of pyridoxal phosphate, the principal cofactor form of vitamin B-6, have achieved new importance with the demonstration that levels of the coenzyme in serum constitute a useful test of vitamin B-6 nutritional status (1). Enzymic assays are among the most satisfactory for measuring pyridoxal phosphate in biological samples for several reasons: 1) they are highly specific, 2) they permit quantification of pyridoxal phosphate at the level of 10−11 moles, and 3) the analyses work well in crude biological extracts without prior isolation or purification.
KeywordsAmmonium Sulfate Pyridoxal Phosphate Potassium Dihydrogen Phosphate Sulfinic Acid Pyruvate Formation
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- 1.Lumeng, L., Ryan, M. P. & Li, T.-K. (1978) Validation of the diagnostic value of plasma pyridoxal 5’-phosphate measurements in vitamin B-6 nutrition of the rat. J. Nutr. 108, 545–553.Google Scholar
- 2.Umbreit, W. W., Bellamy, W. D. & Gunsalus, I. C (1945) The function of pyridoxin derivatives: A comparison of natural and synthetic codecarboxylase. Arch. Biochem. 7, 185–199.Google Scholar
- 3.Haskell, B. E. & Snell, E. E. (1972) An improved apotryptophanase assay for pyridoxal phosphate. Anal. Biochem. 45, 567–576.Google Scholar
- 4.Morino, Y. & Snell, E. E. (1970) Tryptophanase (Escherichia coli B). In: Methods in Enzymology (Tabor, H. & Tabor, C. W., eds.), vol. 17A, pp. 439–447, Academic Press, New York.Google Scholar
- 5.Sloane-Stanley, G. H. (1949) The use of the tyrosine apodecarboxylase of Streptococcus faecalis R for the estimation of codecarboxylase. Biochem. J. 44, 567–573.Google Scholar
- 7.Pardee, A. B. & Prestidge, L. S. (1961) The initial kinetics of enzyme induction. Biochem. Biophys. Acta 49, 77–88.Google Scholar
- 9.McCormick, D. B., Gregory, M. E. & Snell, E. E. (1961) Pyri- doxal phosphokinases. I. Assay, distribution, purification and properties. J. Biol. Chem. 236, 2076–2084.Google Scholar
- 10.Gailani, S. (1965) Pyridoxal phosphate determination in isolated leucocytes and tissue by E. coli apotryptophanse. Anal. Biochem. 13, 19–27.Google Scholar
- 11.Wada, H. & Snell, E. E. (1961) The enzymatic oxidation of pyridoxine and pyridoxamine phosphates. J. Biol. Chem. 236, 2089–2095.Google Scholar
- 12.Bain, J. A. & Williams, H. L. (1970) Concentrations of B-6 vitamers in tissue and tissue fluids. In: Inhibition in the Nervous System and Gamma-aminobutyric Acid (Roberts, E., ed.), pp. 275–293. Macmillan (Pergamon), New York.Google Scholar
- 13.Snell, E. E. (1975) Tryptophanase: Structure, catalytic activities, and mechanism of action. Adv. in Enzymol. 42, 287–333.Google Scholar
- 15.Suelter, C. H., Wang, I. & Snell, E. E. (1976) Application of a direct spectrophotometric assay employing a chromogenic substrate for tryptophanase to the determination of pyridoxal and pyridoxamine 5’-phosphates. Anal. Biochem. 76, 221–232.Google Scholar