CO2+ BINDING TO ALPHA-LACTALBUMIN

alpha-Lactalbumin possesses multiple Zn2+ binding sites, with the stro ngest site having an affinity constant of 5 x 10(5) M(-1) [Permyakov e t al. (1991), J. Protein Chem. 100, 577]. The binding of zinc at secon dary sites is accompanied by destabilization of the protein structure and progressive protein aggregation. This pronounced destabilization i s reflected in a shift of the thermal denaturation transition temperat ure by more than 40 degrees. The present work examines Co2+ binding to bovine alpha-lactalbumin, where for this analog of Zn2+, multiple bin ding sites were also found from spectrofluorimetric titrations. The st rong site Co2+ binding constant was 1.3 x 10(6) M(-1). However, in con trast to Zn2+ binding, Co2+ does not cause protein aggregation nor any significant thermal destabilization of the protein. Fluroescence ener gy transfer measurements between Tb3+ in the strong calcium site to Co 2+ in the strong Zn2+ site gave a distance in the range of 14-18 Angst rom., which was in excellent agreement with recent crystallographic da ta for human cu-lactalbumin [Ren et al. (1993), J. Biol. Chem. 268, 19 292-19298] However, the X-ray structure did not identify the additiona l zinc sites found from earlier solution studies, presumably due to re strictive crystal packing interactions. The results from the current w ork confirm that the strong cobalt (zinc) site in solution is the same zinc site elucidated by X-ray crystallography.