An enzymatic method for determination of B6 vitamers is presented. In this method pyridoxal 5′-phosphate is used to activate aposerine hydroxymethyltransferase to form the catalytically active holoenzyme. The active serine hydroxymethyltransferase, and two other enzymes that form a metabolic cycle, convert serine to glycine and CO2 with the concomitant production of two equivalents of NADPH. The rate of the cycle is directly proportional to the amount of active holoserine hydroxymethyltransferase, which is a measure of the amount of pyridoxal 5′-phosphate in the original sample. The cycle operates about 50 times per minute giving a 100-fold enhancement of NADPH production with respect to original pyridoxal 5′-phosphate content. Other B6 vitamers are converted to pyridoxal 5′-phosphate by a preincubation with a combination of pyridoxal kinase and pyridoxine 5′-phosphate oxidase. A complete analysis of B6 vitamers can be completed in less than 1 h and the assay is linear in the 2- to 50-pmol range of pyridoxal 5′-phosphate. The method is applied to the determination of the B6 vitamer pools in extracts of Escherichia coli. The results show that the pool of pyridoxal 5′-phosphate that is not bound to proteins is large enough to account for product inhibition of both pyridoxal kinase and pyridoxine 5′-phosphate oxidase.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology