A sodium carbonate-bicarbonate buffer for alkaline phosphatases

Abstract

For the study of alkaline phosphatase systems a non-phosphate-containing buffer is required. The buffers in common use for the alkaline pH range are ammonia, borate, glytine and veronal. Ammonia and borate have a retarding effect on the rate of ,enzymic hydrolysis of phosphoric esters. Glycine activates phosphatase when present in very low concentrations (0 1-1 mm), but in the concentrations used in the buffer system (0-01-01M) it has an undoubted inhibitory effect. Veronal is much better in this respect, but its useful pH range (6-8-9-6) is not sufficiently high to cover the pH optima of the enzyme acting on aromatic phosphoric esters (Delory & King, 1943). To satisfy the criteria of non-inhibition of enzymic action, useful alkaline pH range, simplicity and reasonable stability, a mixture of sodium carbonate with bicarbonate has been investigated. The range covered, 9-2-10-8, is suitable for enzymic study with phenolic esters of phosphoric acid. A sodium carbonate buffer was described by Kolthoff (1925), in which a partial transformation into sodium bicarbonate was effected by the addition of hydrochloric acid. We have found it difficult to prepare buffer mixtures in this manner because of the ease with which carbon dioxide can be lost from the solution when the hydrochloric acid is added. The mixtures of sodium carbonate and bicarbonate, on the other hand, are easy to prepare, and have proved to be surprisingly stable. We have kept the mixtures described below for as long as 6 months in well-stoppered waxed bottles without any demonstrable change in their pH.