Studies on proteolytic activity in commercial myoglobin preparations

Area of Science:

• Biochemistry
• Proteomics
• Enzymology

Background:

• Commercial myoglobin preparations are derived from animal skeletal muscle.
• Myoglobin is often associated with other muscle proteins, including enzymes.

Purpose of the Study:

• To investigate the proteolytic activity present in commercial horse skeletal muscle myoglobin preparations.
• To characterize the enzyme responsible for protein degradation and its purification.

Main Methods:

• Proteolytic activity assay using casein and myofibril preparations.
• Ultrafiltration for partial separation of protease from myoglobin.
• Affinity chromatography with immobilized soya-bean trypsin inhibitor for enzyme purification.
• Substrate hydrolysis assays and inhibition studies.

Main Results:

• Commercial myoglobin preparations exhibited significant proteolytic activity against casein and myofibrils, with optimal activity at pH 8-8.5.
• A protease was partially separated from myoglobin via ultrafiltration (MW 30,000 exclusion limit).
• Affinity chromatography yielded a >1000-fold purification of proteolytic activity.
• The purified enzyme hydrolyzed specific ester substrates and was inhibited by trypsin inhibitors, serum, muscle homogenates, EDTA, p-chloromercuribenzoate, and phenylmethylsulphonyl fluoride.

Conclusions:

• Horse skeletal muscle myoglobin preparations contain a potent protease.
• The protease is distinct from myoglobin and can be purified to high levels.
• The enzyme's substrate specificity and inhibition profile suggest it may be a serine protease or metalloprotease, requiring further investigation.