Myoglobin inhibition of most protease activities measured with fluorescentsubstrates is an artifact!

Myoglobin has been suggested to be a potential inhibitor of endogenous musc le proteases as different as cathepsin B, cathepsin L, cathepsin H and calp ains all being supposed to be important in post-mortem muscle. The present work aimed at verifying the ability of myoglobin and its prosthetic group, hemin, to inhibit a series of endopeptidases including papain, cathepsin B, trypsin, calpains as well as two activities of the 20S proteasome. The con clusion of the present work was that inhibition of proteolytic activities o f endopeptidases by myoglobin is an artifact. This was based on the followi ng evidences: (1) a similar extent of inhibition was observed for all prote ases tested whether myoglobin or hemin were added before starting the react ion or after having stopped it; (2) a quenching of the probes fluorescence by myoglobin and hemin; (3) no inhibition of calpains were found when assay ed with non labeled casein as substrate and the activity expressed as the i ncrease in the absorbency at 280 nm of the TCA soluble protein fragments. ( C) 1999 Elsevier Science Ltd. All rights reserved.