Determination of vitamin B₁₂ using the enzyme glycerol dehydrase

Abstract

Glycerol dehydrase is an enzyme that catalyzes dehydration of glycerol into β‐propionaldehyde. It requires 5′‐deoxyadenosylcobalamin, one of the forms of vitamin B₁₂, as a coenzyme. The enzyme is inactivated in vitro by all forms of vitamin B₁₂ stoichiometrically. The objective of this study was to determine vitamin B₁₂ content by utilizing the inactivation of the enzyme by vitamin B₁₂. After various examinations, an excellent standard curve was obtained up to 1 pmol vitamin B₁₂ using 14 mU of the enzyme per tube. Glycerol dehydrase does not respond to vitamin B₁₂ if it is bound to haptocorrin, a vitamin B₁₂‐binding protein. This necessitates a procedure for extraction of vitamin B₁₂ from samples before assay. The enzyme was less inactivated by 5′‐deoxyadenosylcobalamin than any other form of vitamin B₁₂. However, this did not matter because all forms of vitamin B₁₂ were converted into cyanocobalamin during the extraction procedure cited above, which was performed in a buffer containing potassium cyanide.

• Enzymatic assay
• inactivation of GDH by cobalamin
• picomol quantity
• specificity