Kinetics and thermodynamics of the mechanism of interaction of sodium phytate with .alpha.-globulin

Abstract

The precipitation mechanism of alpha-globulin in the presence of myo-inositol hexaphosphate (sodium phytate) was studied in detail. The maximum interaction was found at pH 2.3 where the protein was in a dissociated state having an 8.3S aggregate and a 1.5S monomer. This interaction was predominantly dependent upon the sodium phytate to protein ratio. Velocity sedimentation studies indicated polymer formation due to preferential progressive binding of ligand to polymer, whose size and concentration increased with an increase in sodium phytate concentration. The polymer formation was shown to be ligand mediated and exists independently in solution along with the monomer. The binding isotherm by equilibrium dialysis confirmed differential binding of sodium phytate to the polymer and the monomer as indicated by two sets of binding sites, one having 7 +/- 2 sites of a K value 1.3 x 10(-4) mol-1 and the other having 56 +/- 3 sites with a K value of 2.8 x 10(-3) mol-1. Binding resulted in perturbation of chromophores of protein due to charge effects. The kinetics of the polymer formation was shown to be a pseudo-first-order reaction having two steps. The initial fast reaction involving conformational changes has rate constants of k1 = 52.4 x 10(-3) s-1 and k' = 67.5 x 10(-3) s-1, followed by a slow reaction step of rate constants k2 = 4.3 x 10(-3) s-1 and k'2 = 2.9 x 10(-3) s-1 at sodium phytate concentrations of 1 x 10(-4) M and 5 x 10(-4) M, respectively.

Phytate-degrading enzymes in pig nutrition

“…This complex formation is a bi-phasic reaction, initially phytate binds and changes the conformation of protein in a rapid step and this is followed by a slower, progressive, aggregation of protein and ultimately the protein-phytate complex precipitates when it exceeds a critical size. Rajendran and Prakash (1993) demonstrated this reaction with sodium phytate and α-globulin; protein-phytate complex formation was maximal at pH 2.3 and dependant upon phytate to protein ratios.…”

Protein–phytate interactions in pig and poultry nutrition: a reappraisal

“…It is noteworthy that O'Dell & de Boland (33) detected protein -phytate interactions in soya flakes but not in maize germ or sesame meal. In contrast Kies et al (37) reported protein -phytate interactions in maize at pH 2 and Rajendran & Prakash (27) reported interactions between sesame a-globulin and phytate that peaked at pH 2·3. However, O'Dell & de Boland (33) conducted their studies at pH levels of 4·4 and 9·0, so it appears that pH differences in the in vitro systems account for the apparent discrepancies in outcomes and emphasise the importance of pH to protein -phytate interactions.…”

“…The potential capacity of Ca to disrupt binary protein-phytate interactions may explain the diminished amino acid digestibility responses to supplemental phytase at higher Ca concentrations in the Ravindran et al (42) study. According to the Cosgrove (14) and Rajendran & Prakash (27) descriptions of binary complexes, the likelihood is that phytate is 'protected' by a shield of aggregated protein once complex formation takes place and would be less susceptible to hydrolysis by exogenous phytase. Thus phytase essentially prevents de novo complex formation via the prior hydrolysis of phytate and binary complexes may be an important limiting factor on the extent of enzymic degradation of phytate.…”

Consequences of calcium interactions with phytate and phytase for poultry and pigs

“…At pH levels below the isoelectric point of protein the polyanionic phytate molecule electrostatically binds with residues of the basic amino acids arginine, histidine and lysine. This initial, rapid step is followed by a progressive protein-protein aggregation that may result in precipitation (Rajendran and Prakash, 1993). The crucial proposition is that phytate-bound protein is refractory to pepsin digestion (Camus and Laporte, 1976;Knuckles et al, 1985Knuckles et al, , 1989.…”