Aprotinin stability testing was conducted with Invitrogen Molecular Probes EnzChek® Protease Assay Kit E6639 over a period of 18 weeks. Aprotinin was stored at 4˚C between uses. The activity of trypsin, a protease used to cleave the BODIPY TR-X casein substrate (E6639), was compared to the activity of trypsin with aprotinin (0.6 TIU/ml), a protease inhibitor. Assay plates were read using a fluorescent plate reader with excitation wavelength of 589 nm and emission wavelength of 617 nm.
Aprotinin inhibition of trypsin remains active during the 18 weeks and is relatively stable when the reagent is stored at 4˚C. Data collected over this period indicated the Relative Fluorescence Units (RFU) is inversely correlated to aprotinin activity and shows a slight increase (slightly decreased aprotinin activity) during the 18 weeks. Further testing over the coming months will reveal the stability of aprotinin.
Graph 1. The stability of aprotinin was determined by comparing the activity of aprotinin (0.6 TIU/ml) inhibited trypsin to the proteolytic activity of trypsin (5 ng/ml) on the BODIPY TR-X casein substrate (Invitrogen Molecular Probes EnzChek® Protease Assay Kit E6639). Aprotinin inhibitory activities are inversely correlated to increases in Relative Fluorescence Units (RFU) and are directly proportional to increasing instability.
Graph 2. The inhibitory action of aprotinin (0.6 TIU/ml) on various dilutions of trypsin was compared to the uninhibited proteolytic activity of trypsin on the BODIPY TR-X casein substrate (Invitrogen Molecular Probes EnzChek® Protease Assay Kit E6639). Aprotinin was able to successfully inhibit trypsin at all concentrations, from 20 ng/ml to 312.5 pg/ml, after 18 weeks of storage at 4˚C.