Appraisal of green fluorescent protein as a model substrate for seryl-histidine dipeptide cleaving agent

Summary We have investigated the action of seryl-histidine dipeptide (SH) on Green Fluorescent Protein (GFPxm) by SDS-PAGE and fluorescence techniques. It was found that 1 mmol SH showed a 75 units protease K (PK)-like cleavage effect at 50°C, pH 6.5–7.5. Compared with the results of SH cleavage on BSA, SDS-PAGE experiments showed no obvious fragments resulting from SH cleavage of GFPxm. SH quenched the GFPxm fluorescence greatly prior to cleavage. When the SH concentration reached 150 mM, the fluorescence quenching phenomena stopped. Compared cleavage. When the SH concentration reached 150 mM, the fluorescence quenching phenomena stopped. Compared with seryl-histidine dipeptide, the individual amino acids Ser and His, and these two amino acids mixed together, showed no effects on the spectral characteristics of GFPxm nor cleavage of GFPxm. Further study of the SH protein cleavage mechanism might provide insight for protease mechanisms and biomacromolecule evolution.