Geoffrey W. Mellor

Temperature-dependences of the kinetics of reactions of papain and actinidin with a series of reactivity probes differing in key molecular recognition features

The temperature-dependences of the second-order rate constants (k) of the reactions of the catalytic site thiol groups of two cysteine peptidases papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) with a series of seven 2-pyridyl disulphide reactivity probes (R-S-S-2-Py, in which R provides variation in recognition features) were determined at pH 6.7 at temperatures in the range 4-30 degrees C by stopped-flow methodology and were used to calculate values of DeltaS++, DeltaH++ and DeltaG++. The marked changes in DeltaS++ from negative to positive in the papain reactions consequent on provision of increase in the opportunities for key non-covalent recognition interactions may implicate microsite desolvation in binding site-catalytic site signalling to provide a catalytically relevant transition state. The substantially different behaviour of actinidin including apparent masking of changes in DeltaH++ by an endothermic conformational change suggests a difference in mechanism involving kinetically significant conformational change.

Development and Validation of a Fluoroimmunoassay for Urinary Normetanephrine

The development of a separation fluoriommunoassay for urinary normetanephrine is described. Antiserum specific to normetanephrine was coupled, using cyanogen bromide, to magnetizable cellulose; and fluorescein labelled normetanephrine was synthesized from fluoresceinthiocarbamylethylene diamine and a previously described normetanephrine derivative. Using these reagents it was possible to construct a reproducible standard curve, covering a wide range of concentrations, and to accurately measure the concentration of this metabolite in acid-hydrolysed urine samples. Cross-reactivities of structurally similar compounds were low and the fluoroimmunoassay showed good correlation with an established gas-chromatographic assay. The procedure is rapid; it is possible to accurately determine the normetanephrine concentration of urine samples approximately 2 h after hydrolysis, resulting in an overall assay time of approximately 4 h. This is the first report of a non-isotopic immunoassay for normetanephrine.