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Myoglobin inhibition of most protease activities measured with fluorescent substrates is an artifact!
1Meat Research Station, INRA-Theix, 63122 Saint Genès Champanelle, France.
Abstract:
Myoglobin has been suggested to be a potential inhibitor of endogenous muscle proteases as different as cathepsin B, cathepsin L, cathepsin H and calpains 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 conclusion of the present work was that inhibition of proteolytic activities of endopeptidases by myoglobin is an artifact. This was based on the following evidences: (1) a similar extent of inhibition was observed for all proteases tested whether myoglobin or hemin were added before starting the reaction or after having stopped it; (2) a quenching of the probes fluorescence by myoglobin and hemin; (3) no inhibition of calpains were found when assayed with non labeled casein as substrate and the activity expressed as the increase in the absorbency at 280 nm of the TCA soluble protein fragments.(1).
Insights
Myoglobin and hemin do not inhibit muscle proteases like cathepsin B or calpains. Observed inhibition is an artifact due to probe fluorescence quenching, not actual enzyme inhibition.
Area of Science:
- Biochemistry
- Proteolysis research
- Muscle protein degradation
Background:
- Myoglobin has been proposed as an inhibitor of endogenous muscle proteases, including cathepsin B, cathepsin L, cathepsin H, and calpains.
- These proteases are considered important in post-mortem muscle changes.
Purpose of the Study:
- To investigate the inhibitory potential of myoglobin and its prosthetic group, hemin, on various endopeptidases.
- To determine if myoglobin's effect on protease activity is a genuine inhibition or an artifact.
Main Methods:
- Assayed inhibition of papain, cathepsin B, trypsin, calpains, and 20S proteasome activities.
- Compared inhibition when myoglobin/hemin were added before or after protease reaction.
- Utilized fluorescence quenching assays and casein substrate assays for calpains.
Main Results:
- Myoglobin and hemin showed similar inhibition levels regardless of addition timing (before or after reaction).
- Myoglobin and hemin were found to quench the fluorescence of the probes used in the assays.
- Calpain activity assays using casein substrate showed no inhibition by myoglobin.
Conclusions:
- The observed inhibition of endopeptidases by myoglobin and hemin is an artifact.
- The artifact is likely due to the fluorescence quenching properties of myoglobin and hemin.
- Myoglobin does not inhibit muscle proteases such as calpains.
