Caseins are phosphoproteins and constitute the major protein component of bovine milk. Caseins occur as micelles in the native form, which are kept together by non-covalent interactions between proteins and by calcium phosphate linkages and appear as a highly stabilized dispersion in milk. In order to optimize the chromatographic resolution for a better identification of individual casein fractions, in this work were analyzed the different effects of denaturing solvents and solutions on the structural conformation of caseins. The caseins were obtained from skim milks by precipitation at pH 4.3, and the proteins was dissolved in: water (solution A); 8 M urea in water/acetonitrile (70:30 v/v) (solution B); 0.3% (v/v) β-mercaptoethanol in water/acetonitrile (70:30 v/v) (solution C); 8 M urea in 165 mM Tris-HCl, 44 mM sodium citrate and 0.3% β-mercaptoethanol (solution D). The chromatographic separation of caseins was performed by reversed-phase high-performance liquid chromatographic (RP-HPLC) using a C4 column and the each casein was identified by MALDI-TOF MS. The best chromatographic separation was achieved by treatment of casein powder with solution D. The CD-spectrum of caseins dissolved in this denaturing solution, showed a significant increase in the α-helix conformation and a decrease in the rate of β-sheet, while random coil conformation remained unchanged. This treatment allowed the separation of different casein sub-fractions and the identification of each component of casein portion. References Bonizzi, I., J. N. Buffoni and M. Feligni. 2009. Quantification of bovine casein fractions by direct chromatographic analysis of milk. Approaching the application to a real production context. J. Chromatogr. 1216: 165-168. Bordin, G., Cordeiro Raposo, F., B. de la Calle and A. R. Rodriguez. 2001. Identification and quantification of major bovine milk proteins by liquid chromatography. J. Chromatogr. 928: 63-76. C. Holt. 1992. Structure and stability of bovine casein micelles. Adv Protein Chem. 43: 63-151.
Susceptibility to denaturation of caseins in milk samples for improving protein conformational study and their identification
MANFRA, MICHELE;
2012-01-01
Abstract
Caseins are phosphoproteins and constitute the major protein component of bovine milk. Caseins occur as micelles in the native form, which are kept together by non-covalent interactions between proteins and by calcium phosphate linkages and appear as a highly stabilized dispersion in milk. In order to optimize the chromatographic resolution for a better identification of individual casein fractions, in this work were analyzed the different effects of denaturing solvents and solutions on the structural conformation of caseins. The caseins were obtained from skim milks by precipitation at pH 4.3, and the proteins was dissolved in: water (solution A); 8 M urea in water/acetonitrile (70:30 v/v) (solution B); 0.3% (v/v) β-mercaptoethanol in water/acetonitrile (70:30 v/v) (solution C); 8 M urea in 165 mM Tris-HCl, 44 mM sodium citrate and 0.3% β-mercaptoethanol (solution D). The chromatographic separation of caseins was performed by reversed-phase high-performance liquid chromatographic (RP-HPLC) using a C4 column and the each casein was identified by MALDI-TOF MS. The best chromatographic separation was achieved by treatment of casein powder with solution D. The CD-spectrum of caseins dissolved in this denaturing solution, showed a significant increase in the α-helix conformation and a decrease in the rate of β-sheet, while random coil conformation remained unchanged. This treatment allowed the separation of different casein sub-fractions and the identification of each component of casein portion. References Bonizzi, I., J. N. Buffoni and M. Feligni. 2009. Quantification of bovine casein fractions by direct chromatographic analysis of milk. Approaching the application to a real production context. J. Chromatogr. 1216: 165-168. Bordin, G., Cordeiro Raposo, F., B. de la Calle and A. R. Rodriguez. 2001. Identification and quantification of major bovine milk proteins by liquid chromatography. J. Chromatogr. 928: 63-76. C. Holt. 1992. Structure and stability of bovine casein micelles. Adv Protein Chem. 43: 63-151.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
