PubMed1981
The interaction of urea with lysozyme was studied in the 192-240 nm spectral region by spectrophotometry. The far and middle ultraviolet protein bands records undergo a non-linear "red shift" and "hypochromic effect" under urea titration. These spectral shifts are interpreted basically in terms of decreasing in molar absorptivity due to the binding of the denaturant with the protein chromophores. Two interaction mechanisms with different chromophores involvement are characterized. One of them is noncooperative and its is evidenced by the analysis either of the red shift or of the hypochromic effect showed by the protein far ultraviolet records in urea up to 2 M. This noncooperative effect is represented by two different and independent classes of binding sites in which the tryptophan side chain and the amide peptide bond unit are involved. The calculated stoichiometric constants give the values of 4.61 M-1 for K1 and of 0.078 M-1 for K2, while the site binding constants have the values of 0.852 M-1 for K1 and 0.086 M-1 for K2. The other mechanism which is detected by the middle U.V. band analysis of the protein in urea concentration up to 8 M shows high cooperativity (Hill coefficient of 2.56). Also in this case, tryptophan residues are involved in the binding process. No significant light-scattering influence on absorption measurements is found.