Fractionation and isolation of the multiple forms of prorennin (prochymosin)

Abstract

The heterogeneity of prorennin was studied by chromatography on DEAE-cellulose and microgranular DEAE-cellulose columns, as well as by polyacrylamide-gel electrophoresis. Prorennin prepared by alum treatment, salting-out and chromatography was resolved into three components by a compound gradient of sodium phosphate on microgranular DEAE-cellulose. Polyacrylamide-gel electrophoresis confirmed the chromatographic results, but crystalline rennin was shown to consist of four bands. When prorennin was isolated directly by chromatography, four zymogen components were resolved on microgranular DEAE-cellulose with a modified compound gradient of sodium phosphate. Polyacrylamide-gel electrophoresis confirmed the existence of four multiple forms of prorennin as well as homogeneity of the chromatographic fractions.

Occurrence of Prochymosin Variants in the Abomasum of Bovine Foetuses and Calves

“…Prorennin studied chromatographically showed two components; the activation of each resulted in the formation of chymosin A and B (FOLTMANN, 1970). Later four prochymosins and chymosins were obtained by chromatographic procedure and confirmed by (electrophoretic analysis (ASATO and RAND, 1972;1977). Among four prochymosins /chymosins demonstrated, two forms, A and B have been best determined.…”

“…In order to examine that prochymosin variants are transformed to corresponding chymosins, some extracts were acidified as follows: to 3ml of extracts 0.1 mol/l HCI to pH4.7 was added; the solution was kept at 20°C for 48-60 h. The chymosin is directly formed at p H between 4.5 and 5.5 while the pseudochymosin is the final activation product at p H below 2.5 (PEDERSON et al, 1979). Both active products differ in electrophoretical mobility ( SUZUKI et al, 1989) and direct formation of chymosin is slow (ASATO and RAND, 1977). Acidified extracts were adjusted to p H 6.2 by adding 1 mol/I Na2HP04 before electrophoresis.…”

“…Copyright Clearance Center Code Statement: 0931 -184X/94/4103 -0171$10.00/0 HARBOE, 1992). In recent years numerous investigations of chymosin preparations have revealed a degree of heterogeneity; four components have been obtained by free boundary electrophoresis (ERNSTROM, 1958), polyacrylamide gel electrophoresis (ASATO and RAND, 1971) and isoelectric focusing (DE KONING and DRAAISMA, 1973). Chromatographic studies of cristalline rennin showed three forms: rennin C, B and A in the order of their elution (FOLTMANN, 1970).…”

Milk coagulants

“…All 3 products were formed in the temperature range 5-37 °C (58), the pH range 5-9-7-0 and in up to 1-5 M-NaCl (125), but the relative rates of the 3 reactions varied. From the molecular weights, the amino acid analyses and the products of action of carboxypeptidase A, the 3 components 10 20 H. Arg-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-lle-Val-Glu-Ser-Leu-Ser-Ser-Ser-Glu -I I I I P P P P 30 40 Glu-Ser-lle-Thr-Arg-lle-Asn-Lys-Lys-lle-Glu-Lys-Phe-Gln-Ser-Glu-Glu-Gln-Gln-GIn-P 50 60 Thr-Glu-Asp-Glu-Leu-Gln-Asp-Lys-lle-His-Pro-Phe-Ala-Gln-Thr-Gln-Ser-Leu-Val-Tyr- 70 80 Pro-Phe-Pro-Gly-Pro-lle-Pro-Asn-Ser-Leu-Pro-Gln-Asn-lle-Pro-Pro-Leu-Thr-Gln-Thr- 90 100 were shown to result from progressive hydrolysis from a point close to the C-terminus and were tentatively identified (125) (Fig. 3).…”

Natural heterogeneity of chymosin and pepsin in extracts of bovine stomachs

“…Other authors (Asato & Rand, 1972 have found four forms of (pro)chymosin named A, B, C and D, separated by their electrophoretic mobility. These four components were not well characterised and it is doubtful if they represent different genetic forms.…”