maxima between 390 440 nm the comparison between substrate and product at the same concentration shows dramatically the increase of ten fold in excitation and 6 fold in emission upon enzymatic hydrolysis initial kinetics were monitored at 25 u00b0 with magnetic stirring at optimal conditions excitation 337 nm and emission 410 nm uv spectra the absorbtion spectra of the substrate abz nf 6 and the n terminal product abz thr ile nle oh were recorded on a bechman du 8 spectrophotometer the substrate shows maxima at 328 nm and 254 nm while the cleavage product has maxima at 318 nm and 252 nm fluorescence assay fluorescence measurements on 96 well elisa plates were made with the titertek fluoroskan ii version 3 1 an excitation filter of 355 nm and an emission filter of 430 nm was used ten u03bcl of a stock solution 0 1 mg ml of hiv protease was incubated with five different concentrations of abz nf 6 in a final volume of 100 u03bcl at 37 u00b0 c with the increase in fluorescence monitored in each well every five minutes a stock solution of 1 mm abz nf 6 in dmso was used a standard curve relating changes in fluorescent intensity to changes in concentration of product was used to convert fluorescence changes into molar velocities in order to predetermine the concentration range for k i determination of inhibitors it was found convenient to do a preliminary assay by hplc twenty.