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

The high-affinity calcium-binding sites of bovine and human alpha-lact albumin as well as equine lysozyme were analyzed by Cd-113 NMR spectro scopy, In the case of equine lysozyme, the addition of isotopically en riched 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-lactalbumi n. In addition, a signal upheld of these (delta=-94.7 ppm) was observe d for Cd-113(2+)-substituted human alpha-lactalbumin. The chemical shi fts 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 lysoz yme are somewhat broader than those observed for the EF-hand protein, In addition, the Cd-113 linewidths for the lactalbumins and the lysozy me, especially bovine alpha-lactalbumin, increase dramatically with th e square of the magnetic held 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 Cd2and K+ can compete for occupation of the high-affinity Ca2+-site, Thei r linewidths also to some extent depend on the concentration of the pr otein itself, The Cd-113 NMR results presented here provide further ev idence in favor of a high degree of homology between the Ca2+-binding sites in these functionally diverse proteins, corroborating our earlie r Ca-43 NMR study of these diverse classes of calcium-binding proteins [Aramini et al, (1992) Biochemistry 31, 6761-6768].