Cadmium-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 113Cd NMR spectroscopy. In the case of equine lysozyme, the addition of isotopically enriched 113Cd2+ results in a signal at delta = -75.9 ppm corresponding to the metal ion bound to the lone Ca(2+)-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 upfield of these (delta = -94.7 ppm) was observed for 113Cd(2+)-substituted human alpha-lactalbumin. The chemical shifts of these proteins are in the vicinity of those reported for other Ca(2+)-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 alpha-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 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 113Cd 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 Ca(2+)-site. Their linewidths also to some extent depend on the concentration of the protein itself.(ABSTRACT TRUNCATED AT 250 WORDS)
Aramini
J M
JM
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Hiraoki
T
T
Ke
Y
Y
Nitta
K
K
Vogel
H J
HJ
D000818:Animals; D002104:Cadmium; D002417:Cattle; D006736:Horses; D006801:Humans; D007554:Isotopes; D007768:Lactalbumin / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D015398:Signal Transduction; D017186:Titrimetry
D007554:Isotopes; D002104:Cadmium; D007768:Lactalbumin; D009113:Muramidase