ABSTRACT
A new method of enzyme immobilization has been described using poly(4-methacryloxybenzoic acid) as the carrier. Activation of the polymer, prior to enzyme attachment, was achieved with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The enzyme coupling step proceeded through nucleophilic attack by the protein on a mixed carbonic anhydride. The degree of polymer activation was determined by analysis for quinoline, a by-product of the reaction. The polymer-enzyme complex was compared to the enzyme in solution in terms of pH optimum, substrate kinetics, and thermal denaturation. Potential uses of the polymerenzyme system in chemical synthesis of benzoquinone derivatives are discussed.
Subject(s)
Horseradish Peroxidase/metabolism , Peroxidases/metabolism , Aniline Compounds/pharmacology , Benzoates , Hydrogen Peroxide/pharmacology , Hydrogen-Ion Concentration , Kinetics , Protein Binding , Solubility , Time FactorsABSTRACT
Distribution of 3,2-dimethyl-4-aminobiphenyl (DMAB) and its metabolites in vivo and the metabolism of DMAB by liver in vitro have been studied in the Wistar rat. DMAB-HCI purified by recrystallization and dissolved in ethanol was injected subcutaneously and extractions made from liver, feces, and urine. Similar technical procedures were used to study in vitro metabolism in rat liver homogenates. Two components were isolated from urine and liver having Rf values (thin-layer chromatography) of 0.13 and 0.59, respectively. Three additional metabolites were found in the hydrolyzed fecal fraction. Rechromatography of the major fecal component yielded 6 fluorescent compounds. Gas-liquid chromatography of the most highly fluorescent of these indicated at least 3 additional metabolites. The evidence presented indicates that the liver transforms DMAB to several metabolites which are rapidly transferred in conjugated form to the intestine via the bile. The urine does not appear to be the major excretory route. We have examined the purity of commercially available DMAB free base and DMAB-HCL and found an impurity that comprises approximately 10-24% of the total samle upon GLC analysis, depending upon the supplier. This contaminant was completely removed by recrystallization of the hydrochloride and the chemical identity of purified compound as DMAB confirmed. Recommendations are presented for the use of this purified compound in a biological system.
