ENZYMATIC METHODS FOR THE QUANTIFICATION OF POLYAMINES USING PLANT AMINE OXIDASES

Maize polyamine oxidase, which catalyses specifically the oxidation of spermidine and spermine, and diamine oxidase from Euphorbia characias latex, which reacts only with diamines such as putrescine and cadaverine, have been utilised for developing an enzymatic test for the quantification of di- and polyamines. The enzymatic test was performed exploiting an end-point-assay method, either (1) spectrophotometrically, following the formation of a red-coloured adduct of DELTA-1-pyrroline with ninhydrin reagent, or (2) polarographically, estimating by a Clark electrode the O2 depletion, or (3) spectrofluorimetrically, following the formation of H2O2 by the peroxidase-dependent oxidation of homovanillic acid to a fluorescent product. These methods have a sensitivity ranging between 2 nmoles (fluorimetric method) and 10 nmoles (ninhydrin and polarographic method), and can be applied to the determination of polyamines in crude plant and animal perchloric acid extracts. The polarographic assay was exploited for the determination of acid-soluble polyamines in a perchloric extract of pea or maize stems and mouse liver or brain. The results obtained were compared with those found by thin-layer chromatography.