CD-113 NMR-STUDIES OF BOVINE AND HUMAN ALPHA-LACTALBUMIN AND EQUINE LYSOZYME

The high-affinity calcium-binding sites of bovine and human alpha-lactalbumin as well as equine lysozyme were analyzed by Cd-113 NMR spectroscopy. In the case of equine lysozyme, the addition of isotopically enriched Cd-113(2+) results in a signal at delta=-75.9 ppm corresponding to the metal ion bound to the lone Ca2+-binding site of the protein. A peak at virtually the identical resonance position (delta=-77.1 ppm) was observed in the analogous experiment with bovine alpha-lactalbumin. In addition, a signal upheld of these (delta=-94.7 ppm) was observed for Cd-113(2+)-substituted human alpha-lactalbumin. The chemical shifts of these proteins are in the vicinity of those reported for other Ca2+-binding proteins. The field dependence of the Cd-113 signals for all three proteins and bovine calmodulin were compared. At each field, the Cd-113 signal linewidths for the alpha-lactalbumins and the lysozyme are somewhat broader than those observed for the EF-hand protein. In addition, the Cd-113 linewidths for the lactalbumins and the lysozyme, especially bovine alpha-lactalbumin, increase dramatically with the square of the magnetic field strength, indicative of the presence of nuclear relaxation via chemical shift anisotropy and chemical exchange. The protein-bound Cd-113 signals for the alpha-lactalbumins are also markedly affected by changes in the amount of K+ present, since Cd2+ and K+ can compete for occupation of the high-affinity Ca2+-site. Their linewidths also to some extent depend on the concentration of the protein itself. The Cd-113 NMR results presented here provide further evidence in favor of a high degree of homology between the Ca2+-binding sites in these functionally diverse proteins, corroborating our earlier Ca-43 NMR study of these diverse classes of calcium-binding proteins [Aramini et al, (1992) Biochemistry 31, 6761-6768].