A convenient assay for γ-aminobutyric acid transaminase

Abstract A facile technique is reported for separating succinic semialdehyde dehydrogenase (SSADH) from γ-aminobutyric acid transaminase (GABA-T) in a commercially available mixture of the two enzymes. The dehydrogenase isolated is a suitable coupling enzyme for the spectrophotometric assay of GABA-T from various sources. To obtain SSADH, the enzyme mixture was first passed through a gel filtration column in 50 mM phosphate buffer, pH 7.2, to remove interfering ions. The filtrate was then adsorbed onto an affinity column of commercially obtainable β-NADP-agarose. GABA-T was removed with 50 mM phosphate buffer, pH 7.2. SSADH was then specifically desorbed with 10 mM NADP in the same buffer. A 76% yield was obtained. Contamination by GABA-T and NADPH oxidase was determined to be 0.2 and 0.04%, respectively. The enzyme so prepared was stable, e.g. retaining up to 89% of its activity after 24 days, when stored in concentrated form in the presence of glycerol plus bovine serum albumin or egg yolk lysolecithin. GABA-T activity determined by the coupled spectrophotometric assay was compared to that obtained in a direct radioisotopic method.