RESUMO
1H-NMR spectroscopy and stopped-flow techniques have been used to investigate the binding of a host of metal ions to alpha-lactalbumins from bovine, goat, and human sources. We have identified two 1H-NMR markers diagnostic of metal ion binding to the high-affinity Ca2+-binding site of bovine alpha-lactalbumin, namely the signals corresponding to the delta-CH3 groups of Met-90, and a leucine, tentatively assigned to Leu-96. A number of metal ions other than Ca2+ bind to this site in either slow (La3+, Lu3+, Y3+, Sr2+, Sc3+) or fast (Cd2+, Ba2+, Pb2+) exchange. From competition experiments using this approach, we have determined an affinity series for metal ion binding at this site, in which lanthanides and Y3+ bind the strongest (Y3+>La3+, Lu3+>Ca2+>Sr2+>Cd2+, Pb2+, Ba2+>Sc3+). Several metal ions do not alter the 1H spectrum of bovine alpha-lactalbumin, retaining the protein in an 'apo-like' state. Evidence is given to support the notion that the paramagnetic divalent metal ions Co2+ and Cu2+ bind to a second distinct site, termed the 'zinc site', and that His-68 is involved in metal ion coordination. Finally, stopped-flow techniques using the indicator Xylenol orange were employed to obtain lanthanide off-rates for bovine, human, and goat alpha-lactalbumins (bovine and goat alpha-LA: k(off)(s-1) approximately 0.2 to 0.01 from La3+ to Lu3+; human alpha-LA: k(off)(s-1) approximately 0.02 to 0.001 from La3+ to Lu3+). In each case, we found that k(off) values decreased by an order of magnitude across the series, meaning that the dissociation constants for these metal ions are relatively constant. Data for the bovine and goat proteins are virtually identical, while the off-rates for human alpha-lactalbumin are appreciably slower. In addition, these rates are much slower than the Ca2+ off-rate for the bovine protein (k(off)(s-1) approximately 2 to 5), determined using the fluorescent indicator, BAPTA.
Assuntos
Lactalbumina/química , Espectroscopia de Ressonância Magnética , Metais/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Biomarcadores , Cálcio/metabolismo , Bovinos , Cobalto/metabolismo , Cobre/metabolismo , Ácido Edético/farmacologia , Cabras , Humanos , Concentração de Íons de Hidrogênio , Cinética , Lactalbumina/metabolismo , Metais Terras Raras/metabolismo , Metionina/química , Metionina/metabolismo , Ligação ProteicaRESUMO
In strongly alkaline aqueous KOH solutions containing SiIV in large excess over AlIII, the kinetics of exchange of monomeric silicate with small acyclic aluminosilicate solute species is much more rapid than with either cyclic aluminosilicates or any all-silicate anions. Selective inversion recovery 29Si NMR studies of homogeneous solutions of stoichiometric composition 3.0 mol kg-1 of SiO2, 0.1 mol kg-1 of Al2O3, and 8.0 mol kg-1 of K2O in 60-75% D2O gave rate constants of 2.0 +/- 0.2 kg mol-1 s-1 and 17 +/- 4 s-1 for the forward and reverse reactions of monomeric silicate with (HO)3AlOSiOn(OH)(3-n)(n+1)- (n = 2 or 3) at 0 degree C. These rate constants are more than 10(4)-fold faster than those extrapolated from 60 to 90 degrees C for comparable reactions of silicate anions. The greater lability of acyclic aluminate centers relative to silicate is ascribed partly to the availability of HO- groups for condensation reactions on Al and mainly to the ease of expansion of the coordination number of AlIII beyond 4. The latter attribute is diminished when AlIII is constrained to be tetrahedral in cyclic structures. With respect to the mechanism of formation of zeolites from alkaline aqueous media, it is suggested that small, labile AlOSi units add rapidly to growing zeolitic structures "on demand", whereas the more kinetically inert cage or ring structures cannot. This would explain why a silicate or aluminosilicate structure that is dominant among solute species at equilibrium in the presence of a particular cation may bear little or no geometric relation to the zeolitic framework promoted kinetically by that same cation.