A carbamoyl-transfer reaction has been demonstrated in the urease-catalyzed hydrolysis of urea, using a thymol blue buffer to give a sensitive spectrophotometric measure of acidity changes. This demonstration offers further support for the proposition that carbamate is the first product of the urease-catalyzed hydrolysis of urea. The inhibition of urease by aceto-Jack bean urease (urea amidohydrolase, EC 3.5.1.5) has a cherished place in the enzymologist's heart, since it was responsible for the death blow to the proposition that the protein was merely a carrier of the catalytic species (Northrop, 1961). Nonetheless, the enzyme remains ill understood and is deserving of a detailed and intensive mechanistic attack. Sumner (1951) described urease as "absolutely specific" and until recently only two additional substrates, hydroxyurea and dihydroxyurea, have been

Kinetic, spectral, and other studies establish that hydroxamic acids bind reversibly to active-site nickel ion in jack bean urease. Equilibrium ultracentrifugation studies establish that the molecular weight of native urease is 590 000 +/- 30 000 while that of the subunit formed in 6 M guanidinium chloride in the presence of beta-mercaptoethanol is approximately 95 000. Essentially the same subunit molecular weight (approximately 93 000) is found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent to denaturation in a guanidinium chloride - beta-mercaptoethanol system at various temperatures. Coupled with an equivalent weight of 96 600 for binding of the inhibitors acetohydroxamic acid and phosphoramidate, these results establish securely that urease is a hexamer with one active site per 96 600-dalton subunit. Consistent values for the equivalent weight are obtained by a routine spectrophotometric titration of the active site of freshly prepared urease with trans-cinnamoylhydroxamic acid. General equations are derived which describe spectrophotometric titrations of binding sites of any enzyme with a reversible inhibitor. These equations allow the evaluation of the difference spectrum of the protein-inhibitor complex even when the binding sites cannot readily be saturated with the inhibitor or vice versa.

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Cardiac glycoside poisoning caused by ingestion of plant material is common in tropical and sub-tropical areas. In evaluating the use of the Abbott TDx Digoxin II assay to detect such cases of poisoning, we found it a rapid and convenient method for confirming the ingestion of glycosides from the plants Nerium oleander, Thevetia peruviana, and Adonis microcarpa, and from the toad Bufo marinus. Here we report some clinical cases illustrating our experience with the use of this assay, and describe results of cross-reactivity studies with compounds structurally similar to digoxin. Because of the competitive nature of the immunoassay as well as the complexity of the mixture of cross-reacting cardiac glycosides present in the plant material, the measured apparent digoxin concentration is not linearly related to the cardiac glycoside concentration.

The specificity of ficin has been investigated using a number of ester and peptide substrates. Three families of esters were used: (i) a series of hippuric acid esters, for which the identity of the values suggests the rate-determining deacylation of a hippuryl-ficin intermediate; (ii) a series of iV-acylglycine p-nitrophenyl esters, for which k0S,t/Km was found to be markedly dependent on the size of the nonpolar acyl group, increasing by a factor of 200 in going from the formyl to the ira/w-cinnamoyl derivative; and (iii) a series of yV-benzyloxycarbonylamino acid p-nitrophenyl esters, in which the L-alanine and L-lysine derivatives were the best F JL icin, papain, and bromelain form a group of plant proteinases all of which have an essential active-site sulfhydryl group (Bender and K6zdy, 1965; Glazer and Smith, 1971).