Abstract
Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated β-lactoglobulin and of methylated α-lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated β-casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.
Similar content being viewed by others
REFERENCES
Bertrand-Harb, C., Chobert, J.-M., Dufour, E., and Haertlé, T. (1991). Sci.Aliments 11, 641–652.
Ding, J., Hughes, S. H., and Arnold, E. (1997). Biopolymers 44, 125–138.
Frechet, D., Ehrlich, R., and Remy, P. (1979). Nucleic Acid Res. 7, 1965–1980.
Frister, H., Meisel, H., and Schlimme, E. (1988). Fresenius Z.Anal. Chem. 330, 631–633.
Garat, B. (1999). Biochem.Educ. 27, 232–236.
Halpin, M. I. and Richardson, T. (1985). J.Dairy Sci. 68, 3189–3198.
Henderson, E., Hardin, C. C., Walk, S. K., Tinoco, I., Jr., and Blackburn, E. H. (1987). Cell 51, 899–908.
Hoyne, P. R., Gacy, A. M., McMurray, C. T., and Maher L. J., III (2000). Nucleic Acid Res. 28, 770–775.
Le Doan, T., Perrouault, L., Praseuth, D., Habhoub, N., Decout, J.-L., Thuong, N. T., Lhomme, J., and Hélène, C. (1987). Nucl.Acids Res. 15, 7749–7760.
Leroy, J. L., Gueron, M., Mergny, J-L., and Hélène, C. (1994).Nucleic Acid Res. 22, 1600–1606.
Mailliart, P. and Ribadeau Dumas, B. (1988). J.Food Sci. 53, 743–752.
Manzini, G., Yathindra, N., and Xodo, L. E. (1994). Nucleic Acid Res. 22, 4634–4640.
Mergny, J.-L. and Lacroix, L. (1998). Nucleic Acid Res. 26, 4797–4803.
Mergny, J-L., Phan, A-T., and Lacroix, L. (1998). FEBS Lett. 435, 74–78.
Mills, M., Arimondo, P. B., Lacroix, L, Garestier, T., Hélène, C., Klump, H., and Mergny, J-L. (1999).J.Mol.Biol. 291, 1035–1054.
Moser, H. E. and Dervan, P. B. (1987).Science 238, 645–650.
Pabo, C. O., and Sauer, R. T. (1992). Annu.Rev.Biochem. 61, 1053–1095.
Scaria, P. V., Shire, S. J., and Shafer, R. H. (1992). Proc.Natl.Acad. Sci.USA 89, 10336–10340.
Sitohy, M. Z., Chobert, J.-M., and Haertlé, T. (2000). J.Food Biochem. 24, 381–398.
Sitohy, M. Z., Chobert, J.-M., and Haertlé, T. (2001).Int.Dairy J. 11, 875–885.
Wintjens, R., Liévin, J. Rooman, M., and Buisine, E. (2000). J.Mol. Biol. 302, 395–410.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sitohy, M., Chobert, JM., Schmidt, M. et al. Interactions Between Esterified Whey Proteins (α-Lactalbumin and β-Lactoglobulin) and DNA Studied by Differential Spectroscopy. J Protein Chem 20, 633–640 (2001). https://doi.org/10.1023/A:1013716202650
Published:
Issue Date:
DOI: https://doi.org/10.1023/A:1013716202650