Gerald L. Rowley

Homogeneous enzyme immunoassay for proteins employing β-galactosidase

Abstract A homogeneous enzyme immunoassay method for proteins is described. The assay components include a protein antigen—β-galactosidase conjugate, antiprotein antibody, and a synthetic macromolecular substrate. When antibody binds to the conjugate, enzyme activity is inhibited up to 95% because of steric exclusion of the substrate. Free-protein antigen competes for antibody, thus preventing inhibition. The extent of inhibition is related to the concentration of protein analyte. The assay is simple to perform, highly sensitive, and is generally useful for proteins with wide structural variations.

PEI-cellulose thin-layer chromatography product studies of the creatine kinase and pyruvate kinase reactions

Abstract Detailed methods for polyethylenimine-cellulose thin-layer chromatographic determination of the reactants and products of the creatine kinase and pyruvate kinase reactions are presented. The technique has been developed as a highly sensitive method both for estimation of relative initial rates afforded by creatine and creatine analogs in the creatine kinase reaction and also for estimation of trace amounts of inorganic phosphate and ADP contamination in samples of ATP.

A method for controlled coupling of amino compounds to enzymes: A homogeneous enzyme immunoassay for gentamicin

Abstract A new method for coupling of amino compounds to enzymes is described. An aminoglycoside, gentamicin, is functionalized with a nucleophilic sulfhydryl group on its deoxystreptamine ring and an enzyme, glucose 6-phosphate dehydrogenase (G6PDH), is modified with electrophilic bromoacetylglycyl groups. Upon modification, the G6PDH loses about 70% of its enzyme activity. Coupling of the modified G6PDH with the sulfhydryl-substituted gentamicin affords, without further loss in activity, the drug-enzyme conjugate. Gentamicin antibodies inhibit up to 76% of the enzyme activity of the conjugate. The conjugate is demonstrated to be useful for a sensitive, homogeneous enzyme immunoassay for gentamicin in human serum.

Action of β-galactosidase on novel synthetic macromolecular substrates. A processive enzymic reaction controlled by coulombic interactions☆

Macromolecular beta-galactosidase substrates were prepared by attaching o-nitrophenyl-beta-galactoside to carboxymethyldextran with positively charged linking groups. Almost all of the substituents were susceptible to enzymic hydrolysis by two distinct pathways. Under some conditions, there was random reaction to give a soluble product. In other conditions, in the initial stages of the reaction, most of the substituents of some, but not all, of the substrate polymers were hydrolyzed to give a product which precipitated as a second aqueous phase. Kinetics of hydrolysis were studied with respect to charge and molecular weight of both the enzyme and substrate. Factors that caused a decrease in Km favored formation of the second phase product. The reaction has similarities to the processive catalytic reactions found in naturally occurring enzyme systems with polymeric charged substrates.