A functional comparison of ovine and porcine trypsins.
Trypsin was isolated from ovine and porcine pancreas using affinity chromatography on immobilized p-aminobenzamidine. Molecular masses of the two proteins were 23900 and 23435 Da, determined by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry. The purified trypsins were compared using the kinetic properties K(m) and k(cat) which were determined at pH 8.0 and between 25 and 55 degrees C. Comparison of the Michaelis constants for ovine and porcine trypsins toward N-alpha-benzoyl-arginine-p-nitroanilide (BapNA) indicated that ovine trypsin had higher affinity for this substrate than the porcine enzyme. The rates of the reactions catalysed by the two enzymes correlated strongly over the range of temperatures and substrate concentrations tested, as did the k(cat) values. The specific activity of ovine trypsin for BapNA was, on average, approximately 10% higher than that of the porcine enzyme over the range of conditions tested. Porcine trypsin was less susceptible to denaturation at low pH or high temperature than was ovine trypsin. Porcine and ovine trypsin produced seven identically sized fragments from auto-catalytic hydrolysis. Proposed regions of identity between ovine and porcine trypsins were I(54)-K(77), L(98)-R(107), S(134)-K(178) and N(209)-K(116). Hydrolysis of beta-lactoglobulin, egg white lysozyme or casein by ovine or porcine trypsin yielded virtually identical patterns of fragments although the rate at which fragments were produced, in the case of beta-lactoglobulin, differed between the two enzymes. On balance the two enzymes appear to be functionally identical in their action.
Dallas Johnson
Keryn
K
School of Biological Sciences, Victoria University of Wellington, P.O. Box 600, Wellington, New Zealand.
Clark
Alan
A
Marshall
Sue
S
D000818:Animals; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000378:metabolism; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D012756:Sheep / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine / Q000502:physiology*; D014357:Trypsin / Q000302:isolation & purification / Q000502:physiology*
D007782:Lactoglobulins; D014357:Trypsin