Cadmium-113 NMR Studies of Bovine and Human a-Lactalbumin and Equine Lysozyme1

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Abstract
The high-affinity calcium-binding sites of bovine and human α-lactalbumin as well as equine lysozyme were analyzed by 113Cd NMR spectroscopy. In the case of equine lysozyme, the addition of isotopically enriched 113Cd2+ results in a signal at δ= − 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 (δ= −77.1 ppm) was observed in the analogous experiment with bovine α-lactalbumin. In addition, a signal upfield of these (β =−94.7 ppm) was observed for 113Cd2+-substituted human α-lactalbumin. The chemical shifts of these proteins are in the vicinity of those reported for other Ca2+-binding proteins. The field dependence of the 113Cd signals for all three proteins and bovine calmodulin were compared. At each field, the 113Cd signal linewidths for the α-lactalbumins and the lysozyme are somewhat broader than those observed for the EF-hand protein. In addition, the 113Cd linewidths for the lactalbumins and the lysozyme, especially bovine α-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 113Cd signals for the α -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 113Cd 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 43Ca NMR study of these diverse classes of calcium-binding proteins.

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