In the present paper, the interaction between copper ions and proteins is presented, in order to elaborate a simple and rapid spectrophotometric assay of casein in milk. Under alkaline conditions, copper ions form the biuret complex with the proteins, which can be used in protein determination. Although very specific, the biuret method is less sensitive. Using insoluble copper phosphate, casein is able to extract copper ions, with which it forms the biuret complex, while either the complex or copper ions could be determined in the ultraviolet range. Indeed, an increased absorbance of biuret complex at 215 nm was found. Nevertheless, copper ions can be determined in UV as well, their concentration being proportional to that of casein. When used tetraglycine instead casein, mass spectrometric measurements at pH higher than 11 revealed the formation of complexes with many copper ions bound to each peptide bond-containing molecule. Nevertheless, on diluting the biuret solution the complex may dissociate leading to very complex UV spectra that should be further studied.
1. Kamizake, N. K.; Gonçalves, M. M.; Zaia, C. T; Zaia, D. A. Determination of total proteins in cow milk powder samples: a comparative study between the Kjeldahl method and spectrophotometric methods. J. Food Comp. Anal.2003, 16, 507-516.
2. Laporte, M. F.; Paquin, P. Near-infrared analysis of fat, protein, and casein in cow's milk. J. Agric. Food Chem.1999, 47, 2600-2605.
3. Lüthi-Peng, Q.; Puhan, Z. Determination of protein and casein in milk by fourth derivative UV spectrophotometry. Anal. Chim. Acta1999, 393, 227-234.
4. Nakai, S.; Le, A. C. Spectrophotometric determination of protein and fat in milk simultaneously. J. Dairy Sci.1970, 53, 276-278.
5. Etzion, Y.; Linker, R.; Cogan, U.; Shmulevich, I. Determination of protein concentration in raw milk by mid-infrared Fourier transform infrared/attenuated total reflectance spectroscopy. J. Dairy Sci.2004, 87, 2779-2788.
6. Barbano, D. M.; Dellavalle, M. E. Rapid method for determination of milk casein content by infrared analysis. J. Dairy Sci.1987, 70, 1524-1528.
7. Ashworth, U. S.; Seals, R.; Erb, R. E. An improved procedure for the determination of milk proteins by dye binding. J. Dairy Sci.1960, 43, 614-623.
8. Kuaye, A. Y. An ultraviolet spectrophotometric method to determine milk protein content in alkaline medium. Food Chem.1994, 49, 207-211.
9. Keller, R. P.; Neville, M. C. Determination of total protein in human milk: comparison of methods. Clin. Chem.1986, 32, 120-123.
10. Albanell, E.; Caja, G.; Such, X.; Rovai, M.; Salama, A. A.; Casals, R. Determination of fat, protein, casein, total solids, and somatic cell count in goat's milk by near-infrared reflectance spectroscopy. J. AOAC Intern.2003, 86, 746-752.
11. Luginbühl, W. Evaluation of designed calibration samples for casein calibration in Fourier transform infrared analysis of milk. LWT-Food Sci. Technol.2002, 35, 554-558.
12. Webster, G. C. Comparison of direct spectrophotometric methods for the measurement of protein concentration. Biochim. Biophys. Acta (BBA)-Protein Struct.1970, 207, 371-373.
13. Kamishikiryo-Yamashita, H. I. R. O. M. I.; Oritani, Y.; Takamura, H; Matoba, T. Protein content in milk by near-infrared spectroscopy. J. Food Sci.1994, 59, 313-315.
14. Gornall, A. G.; Bardawill, C. J.; David, M. M. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem, 1949, 177, 751-766.
15. Gradinaru, R.; Murariu, M.; Dragan, E. S.; Drochioiu, G. Protein determination based on the biuret absorbtion in the UV range and copper binding to peptides. Rom. Biotech. Lett., 2007,12, 3235-3239.
16. Drochioiu, G.; Damoc, E. N.; Przybylski, M. Novel UV assay for protein determination and the characterization of copper-protein complexes by mass spectrometry. Talanta2006, 69, 556-564.
17. Schlosser, G.; Stefanescu, R.; Przybylski, M.; Murariu, M.; Hudecz, F.; Drochioiu, G. Copper-induced oligomerization of peptides: a model study. Eur. J. Mass Spectrom. 2007, 13, 331-337.
18. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.1976, 72, 248-254.