Isolation and characterization of pepsin from polar cod (Boreogadus saida)

1. 1. Two zymogens of pepsin, pepsinogens A and B, were isolated from the gastric mucosa from Polar cod. Pepsins A and B accounted for more than 90% of the total pepsin units obtained in the crude extract of the mucosa. 2. 2. Pepsinogen A had a mol. wt of 42,000 and a P1 of 3.75. With hemoglobin as substrate, pepsin A had pH optimum of 2.0, a Km′ of 0.06 mM, a temp optimum of 37°C, an Arrhenius activation energy of 3.2 kcal/mol, and a physiological efficiency of 517. 3. 3. Pepsinogen B had a mol. wt of 40,000 and a P1 of 4.75. Pepsin B, with hemoglobin as substrate, had a pH optimum of 2.0, a temperature optimum of 37°C, a Km′ of 1.33 mM, an Arrhenius activation energy of 2.9 kcal/mol and a physiological efficiency of 418. 4. 4. Pepsins A and B clotted milk when the substrate pH was below 6.6. The temperature coefficient of milk clotting was 1.2–1.4 compared to 2–2.2 for pepsins from mammals. 5. 5. Under standard milk clotting conditions, pH 6.2 and 30°C, the milk clotting activity declined more rapidly as enzyme concentration was reduced more than would be expected. 6. 6. The non-linearity of the enzyme dilution curve was caused by the instability of Polar cod pepsin at pH 6.2, 30°C.

[1]  H. Smit.  Influence of temperature on the rate of gastric juice sectretion in the brown bullhead, Ictalurus nebolosus , 1967 .

[2]  A. Allen.  Mucus — a protective secretion of complexity , 1983 .

[3]  C C Bigelow,et al.  On the average hydrophobicity of proteins and the relation between it and protein structure. , 1967, Journal of theoretical biology.

[4]  S. Colowick,et al.  Methods in Enzymology , Vol , 1966 .

[5]  K. Weber,et al.  The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. , 1969, The Journal of biological chemistry.

[6]  N. Haard,et al.  Modification of Proteins with Proteolytic Enzymes from the Marine Environment , 1982 .

[7]  E. Hofmann,et al.  An improved procedure for protein staining in polyacrylamide gels with a new type of Coomassie Brilliant Blue. , 1972, Analytical biochemistry.

[8]  B. Davis.  DISC ELECTROPHORESIS – II METHOD AND APPLICATION TO HUMAN SERUM PROTEINS * , 1964, Annals of the New York Academy of Sciences.

[9]  G. Parker.  General Anesthesia by Cooling , 1939 .

[10]  T. G. Owen,et al.  Thermal compensation in the stomach of the brook trout (Salvelinus fontinalis Mitchill) , 1971 .

[11]  W. Taylor.  Proteinases of the stomach in health and disease. , 1962, Physiological reviews.

[12]  J. Field,et al.  The respiratory metabolism of excised tissues of warm- and cold-adapted fishes. , 1950, The Biological bulletin.

[13]  A. V. Hooydonk,et al.  Derivation of a mathematical model for the mechanism of casein micelle coagulation by rennet , 1981, Journal of Dairy Research.

[14]  P. Low,et al.  Temperature adaptation of enzymes: roles of the free energy, the enthalpy, and the entropy of activation. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[15]  A. Mirsky,et al.  THE ESTIMATION OF PEPSIN WITH HEMOGLOBIN , 1932, The Journal of general physiology.

[16]  K. Fujiwara,et al.  Affinity chromatography of several proteolytic enzymes on carbobenzoxy-D-phenylalanyltriethylenetetramine-sepharose. , 2009, International journal of peptide and protein research.

[17]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[18]  M. Pollock.  PURIFICATION AND PROPERTIES OF PENICILLINASES FROM TWO STRAINS OF BACILLUS LICHENIFORMIS: A CHEMICAL, PHYSICOCHEMICAL AND PHYSIOLOGICAL COMPARISON. , 1965, The Biochemical journal.