Determination of serum lactate with alkylamine glass bound lactate oxidase

Abstract

Commercial lactate oxidase (Lactate:O₂; oxidoreductase EC 1.1.3.2) from Pediococcus species was immobilized on to al-kylamine glass beads (pore diameter 55 nm) through glutaralde-hyde coupling with a conjugation yield of 3.2 mg/g support and 105% retention of initial activity. Immobilized enzyme showed maximum activity at pH 6.5, when incubated at 40ºC for 12 min and was used for determination of lactic acid in serum. The H₂O₂ generated from serum lactate by immobilized enzyme was meas-ured colorimetrically at 565 nm by its oxidative coupling with 4-aminoantipyrine and N,N'-dimethyaniline catalyzed by horse-radish peroxidase. A linear relationship was observed between A₅₆₅ and lactic acid concentration ranging from 0.075 mM to 10 mM. The minimum detection limit of the method was 0.075 mM, which was better than that of enzymic colorimetric method employing free enzyme (0.2 mM). Within day and between day coefficient of variations were <8.0% and <19%, respec-tively. Serum lactic acid values determined by the present method were in good correlation (r = 0.99) with the currently used enzymic colorimetric method. The cost of lactate determination for 100 serum samples was less, as compared with Sigma kit method.