Novel route for agmatine catabolism in Aspergillus niger: 4-guanidinobutyrase assay

Abstract

The enzyme 4-guanidinobutyrase (GBase) catalyzes the hydrolysis of 4-guanidinobutyric acid (GB) to 4- aminobutyric acid (GABA) and urea. Here we describe methods to estimate urea and GABA that were suitably adapted from the published literature. The urea is determined by colorimetric assay using modified Archibald’s method. However, the low sensitivity of this method often renders it impractical to performfine kinetic analysis. To overcome this limitation, a high sensitive method for detecting GABA is exploited that can even detect 1 μM of GABA in the assay mixture. The samples are deproteinized by perchloric acid (PCA) and potassium hydroxide treatment prior to HPLC analysis of GABA. The method involves a precolumn derivatization with o-phthalaldehyde (OPA) in combination with the thiol 3-mercaptopropionic acid (MPA). The fluorescent GABA derivative is then detected after reversed phase high performance liquid chromatography (RP-HPLC) using isocratic elution. The protocols described here are broadly applicable to other biological samples involving urea and GABA as metabolites.