Bovine metalloprotease characterization and in vitro connective tissue degradation.
Metalloproteases that selectively hydrolyze connective tissue proteins may tenderize meat without creating texture problems associated with myofibrillar protein degradation. Our objective was to characterize the activity of bovine placental proteases to determine whether they can improve meat tenderness through disruption of the connective tissue matrix. Enzymes were extracted, crudely purified, and proteolytic activity was assessed against gelatin and collagen under varying pH and temperature conditions using both SDS-PAGE and zymography. Gelatin zymography revealed proteolysis between 57 and 63 kDa, with decreased activity as buffer pH decreased from pH 7.4 to 5.4 (37 degrees C). Proteolytic activity was pronounced at 37 degrees C, moderate at 25 degrees C, and absent at 4 degrees C following 48-h incubation (pH 7.4). Placental enzymes were metalloproteases inhibited by excess EDTA. Maximum proteolysis was achieved in the presence of Ca2+, with or without Mg2+ and Zn2+. Absence of Ca2+ decreased proteolytic activity. Complete degradation of both the 125- and 120-kDa proteins of the alpha-chains of gelatin was achieved following enzyme incubation for 6 h at 37 degrees C or 24 h at 25 degrees C. No degradation was observed following enzyme incubation with native Type I collagen. Given the marked decrease in enzyme activity at pH 5.4 and 4 degrees C (standard industry conditions), bovine placental metalloproteases would not be expected to contribute to connective tissue degradation or improve meat tenderness.
Alderton
A L
AL
Department of Animal and Food Sciences, University of Kentucky, Lexington 40546, USA.
Means
W J
WJ
Kalchayanand
N
N
McCormick
R J
RJ
Miller
K W
KW
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003094:Collagen / Q000378:metabolism; D003238:Connective Tissue / Q000187:drug effects / Q000378:metabolism*; D046508:Culture Techniques / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005511:Food Handling / Q000379:methods*; D005780:Gelatin / Q000378:metabolism; D005782:Gels; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008460:Meat / Q000592:standards*; D045726:Metalloproteases / Q000378:metabolism* / Q000494:pharmacology; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology; D011506:Proteins / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature
D005782:Gels; D011506:Proteins; D005780:Gelatin; D003094:Collagen; D045726:Metalloproteases; D002118:Calcium