Co2+ binding to alpha-lactalbumin.
alpha-Lactalbumin possesses multiple Zn2+ binding sites, with the strongest site having an affinity constant of 5 x 10(5) M-1 [Permyakov et al. (1991), J. Protein Chem. 100, 577]. The binding of zinc at secondary sites is accompanied by destabilization of the protein structure and progressive protein aggregation. This pronounced destabilization is reflected in a shift of the thermal denaturation transition temperature by more than 40 degrees. The present work examines Co2+ binding to bovine alpha-lactalbumin, where for this analog of Zn2+, multiple binding sites were also found from spectrofluorimetric titrations. The strong site Co2+ binding constant was 1.3 x 10(6) M-1. However, in contrast to Zn2+ binding, Co2+ does not cause protein aggregation nor any significant thermal destabilization of the protein. Fluorescence energy transfer measurements between Tb3+ in the strong calcium site to Co2+ in the strong Zn2+ site gave a distance in the range of 14-18 A, which was in excellent agreement with recent crystallographic data for human alpha-lactalbumin [Ren et al. (1993), J. Biol. Chem. 268, 19292-19298]. However, the X-ray structure did not identify the additional zinc sites found from earlier solution studies, presumably due to restrictive crystal packing interactions. The results from the current work confirm that the strong cobalt (zinc) site in solution is the same zinc site elucidated by X-ray crystallography.
Permyakov
E A
EA
Department of Chemistry, Ohio State University, Columbus 43210.
Berliner
L J
LJ
D000817:Anilino Naphthalenesulfonates; D002118:Calcium / Q000494:pharmacology; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000378:metabolism*; D011485:Protein Binding; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D013725:Terbium; D003035:Cobalt; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D007768:Lactalbumin; D002118:Calcium