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A method for determination of macropeptide by cation-exchange fast protein liquid chromatography and its use for following the action of chymosin in milk

Published online by Cambridge University Press:  01 June 2009

Joëlle Léonil  and
Daniel Mollé
Joëlle Léonil
Affiliation:
Laboratoire de Recherches de Technologie Laitière, INRA, 65 rue de Saint-Brieuc, 35042 Rennes Cédex, France
Daniel Mollé
Affiliation:
Laboratoire de Recherches de Technologie Laitière, INRA, 65 rue de Saint-Brieuc, 35042 Rennes Cédex, France
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Summary

Cation-exchange chromatography on a Mono S column (Pharmacia) was used to separate macropeptide from whey proteins. Macropeptide was eluted by 0·1 M-NaCl in a 20 mM-KCl–HCl buffer, pH 2. This technique was suitable for quantitative determination of macropeptide in rennet whey and also for following the action of chymosin on κ-casein in skim milk. Precipitation at pH 4·6 was used to remove residual caseins and to keep macropeptide in solution. In comparison with other methods for determining macropeptide, the present one eliminates the need for pretreatment of samples with trichloroacetic acid (TCA) and allows the recovery of all the macropeptide. Quantitative determination of macropeptide in the 8% TCA-soluble fraction by cation-exchange chromatography showed that only 50–75% of the macropeptide was recovered. This chromatographic technique could also be applied for isolating and producing whole macropeptide on a preparative scale.

Type
Original Articles
Information
Journal of Dairy Research , Volume 58 , Issue 3 , August 1991 , pp. 321 - 328
Copyright
Copyright © Proprietors of Journal of Dairy Research 1991

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References

REFERENCES

Andrews, A. T. 1983 Proteinases in normal bovine milk and their action on caseins. Journal of Dairy Research 50 45–55CrossRefGoogle ScholarPubMed
Armstrong, C. E., MacKinlay, A. G., Hill, R. J. & Wake, R. G. 1967 The action of rennin on k-casein: the heterogeneity and origin of the soluble product. Biochimica et Biophysica Acta 140 123–131CrossRefGoogle Scholar
Carles, C. & Martin, P. 1985 Kinetic study of the action of bovine chymosin and pepsin A on bovine k-casein. Archices of Biochemistry and Biophyaics 242 411–416CrossRefGoogle Scholar
Chaplin, B. & Green, M. L. 1980 Determination of the proportion of k-casein hvdrolysed by rennet on coagulation of skim-milk. Journal of Dairy Research 47 351–358CrossRefGoogle Scholar
Dalgleish, D. G. 1979 Proteolysis and aggregation of casein micelles treated with immobilized or soluble chymosin. Journal of Dairy Research 46 653–661CrossRefGoogle Scholar
De Koning, P. J., Jenness, R. & Wijnand, H. P. 1963 Some aspects of the determination of N-acetyl neuraminic acid, and its use for the comparison of the action of heat and rennin on casein. Netherlands Milk and Dairy Journal 17 352–363Google Scholar
Delfour, A., Jolles, J., Alais, C. & Jolles, P. 1905 Caseino-glycopeptides: characterization of a inethionine residue and of the N-terminal sequence. Biochemical and Biophysical Research Communications 19 452–455CrossRefGoogle Scholar
Fox, K. K., Holsinger, V. H., Posati, L. P. & Paliansch, M. J. 1967 Separation of β-lactoglobulin from other milk serum proteins by trichloroacetic acid. Journal of Dairy Science 50 1363–1367CrossRefGoogle Scholar
Fox, P. F. 1909 Milk-clotting and proteolytic aetivities of rennet. and of bovine pepsin and porcine pepsin. Journal of Dairy Research 36 427–433CrossRefGoogle Scholar
Léonil, J. & Mollé, D. 1990 Liberation of tryptic fragments from caseinomacropeptide of bovine k-casein involved in platelet function. Kinetic study. Biochemical Journal 271 247–252CrossRefGoogle ScholarPubMed
Mercier, J.-C., Uro, J., Ribadeau, Dumas B.Grosclaude, F. 1972 [Primary structure of the caseinomacropeptide of bovine k-casein B1.] European Journal of Biochemistry 27 535–547CrossRefGoogle Scholar
Olieman, C. & Van Den Bedem, J. W. 1983 A sensitive HPLC method of detecting and estimating rennet whey total solids in skim milk powder. Netherlands Milk and Dairy Journal 37 27–36Google Scholar
Olieman, C. & Van Riel, J. A. M. 1989 Detection of rennet whey solids in skim milk powder and buttermilk powder with reversed-phase HPLC. Netherlands Milk and Dairy Journal 43 171–184Google Scholar
Pearce, K. N. 1979 Use of fluorescamine to determine the rate of release of the caseino-macropeptide in rennet-treated milk. New Zealand Journal of Dairy Science and Technology 14 233–239Google Scholar
Sinkinson, G. & Wheelock, J. V. 1970 Carbohydrates of the glycopeptides released by the action of rennin on whole milk. Biochimica et Biophysica Acta 215 517–521CrossRefGoogle ScholarPubMed
Swaisgood, H. E. 1982 Chemistry of milk protein. In Developments in Dairy Chemistry 1. Proteins pp. 159 (Ed. Fox, P. F.). London: Applied Science PublishersGoogle Scholar
Van Hooydonk, A. C. M. & Olieman, C. 1982 A rapid and sensitive high-performance liquid chromatography method of following the action of chymosin in milk. Netherlands Milk and Dairy Journal 36 153–158Google Scholar
Vreeman, H. J., Visser, S., Slangen, C. J. & Van Riel, J. A. M. 1986 Characterization of bovine k-casein fraction and the kinetics of chymosin-induced macropeptide release from carbohydrate-free and carbohydrate-containing fractions determined by high-performance gel-permeation chromatography. Biochemical Journal 240 87–97CrossRefGoogle ScholarPubMed
Warren, L. 1959 The thiobarbituric acid assay of sialic acids. Journal of Biological Chemistry 177 751–766Google Scholar
Wheelock, J. V. & Knight, D. J. 1969 The action of rennet on whole milk. Journal of Dairy Research 36 183–190CrossRefGoogle Scholar
Yvon, M., Chabanet, C. & Pélissier, J.-P. 1989 Solubility of peptides in trichloroacetic acid (TCA) solutions. Hypothesis on the precipitation mechanism. International Journal of Peptide and Protein Research 34 166–176CrossRefGoogle ScholarPubMed