A RAPID HOMOGENEOUS FLUORESCENCE ASSAY FOR SUBTILISIN

A rapid and sensitive homogeneous assay method has been developed for the determination of subtilisin. The method employs a protein substrat e labelled with two fluorescent dyes with fluorescence energy transfer (FET) characteristics. The doubly-labelled substrate was prepared by chemically coupling bovine serum albumin with lucifer yellow and rhoda mine dyes. The fluorescence emission from the lucifer labels was initi ally quenched due to the FET to the adjacent rhodamine labels. However , upon the addition of subtilisin into the labelled substrate solution , increased fluorescence was observed as the enzyme hydrolyzed the sub strate and reduced the FET effect. The rate of increase in fluorescenc e due to substrate hydrolysis was used to calibrate the subtilisin ass ay. It was linear over the range 0-150 ng of the enzyme (n = 8, r(2) = 0.985). The assay was fast with a time of 30 sec to exceed the limit of detection (LOD) signal for 60 ng of subtilisin in 600 mu l. In this volume, the LOD for the enzyme was 4.2 ng (99% confidence).