Charles J. Martin

Phytic acid:divalent cation interactions. V. titrimetric, calorimetric, and binding studies with cobalt(ii) and nickel(ii) and their comparison with ot

Abstract The ionization characteristics and accompanying heat changes of phytic acid ( myo -inositol hexaphosphate) in the presence of varying concentrations of Co(II) and Ni(II) have been studied over the pH range 2.5–10.5 in the presence of 0.20 M KNO 3 at 25°C. The effect of the binding of these metal ions on the ionization constants of the ionizable phosphate groups is qualitatively similar to that seen with the cations of Zn(II), Cu(II), and Ca(II), viz., displacement of the phytic acid titration curve to more acid pH values. Quantitatively, the overall effect of Co(II) and Ni(II) binding on the ionization constants of phytic acid is greater than that induced by the interaction with Ca(II) but less than that observed by the binding of Zn(II) or Cu(II) to phytate. The effect of pH on the binding of Ni(II) to phytate has been determined spectrophotometrically and compared with results obtained for the binding of Co(II), Cu(II), and Ca(II) to phytate. The binding of Ni(II) occurs over the pH range from ca. 5 to 8.2 and with the formation of insoluble complexes. Evidence for the formation of soluble complexes was not observed. This contrasts with the results obtained with Cu(II) and Co(II), where soluble complexes with phytate are found at pH 2 to ca. 3. However, in conformity with the results obtained with these two metal ions, it appears that 6 mois Ni(II) can bind to 1 mol phytate. From previous studies, the maximal binding ratio of Ca(II): phytate was found to be 5; for Zn(II), it appears to be only from 3.5 to 4. On the basis of pH binding profiles, it is proposed that the binding of Cu(II), Co(II), and possibly Zn(II) occurs to the equatorial configuration of phytate. The axial form, existing in solution above pH 5, is probably the form to which both Ca(II) and Ni(II) bind. A comparison of the heat changes associated with the deprotonation of phytic acid with those measured in the presence of divalent cations permits evaluation of an apparent heat of binding term For the systems studied, this endothermic heat change (22–27 kcal mol −1 ) appears to be little affected by either the type of metal ion or the extent of binding Although there are multiple contributors to these heat of binding values, it is suggested that among them the heat of precipitation is probably the largest and effectively blankets heat changes due to the specific binding effects The titration behavior of the aqud metal ions and the heat changes that arise as a function of pH during their conversion to their hydroxides have also been studied Their acidity constants (p K * ) were found to be in the increasing order of Cu(II) > Zn(II) > Ni(II) > Co(II) with the exothermic ΔH° values varying from − 7 5 kcal mol −1 for Ni(II) to − 12 8 kcal mol −1 for the titration of Cu(II) Values of ΔG° calculated from the acidity constants (p K * can be equated to −log K ) permitted determination of ΔS° Changes in the entropy varied from − 62 to − 72 e.u. As has been found for the ionization of many compounds (phenols, carboxylic acids, etc ) a linear relationship was found to exist between ΔH° and ΔG° and therefore between ΔH° and ΔS°. The solubility of metal lon-phytate complexes as a function of pH at various mol ratios has been studied by the measurements of the apparent absorbance at 400 nm Under conditions wherein pH hysteresis effects are observed, time-dependent changes in “absorbance” also occur rhe two phenomena are not always synchronous processes. At metal ion phytate mol ratios of 6, kinetics of the pH hysteresis effects were such that the biphasic rate curves could be fitted to an equation descriptive of the simultaneous occurrence of two first order reactions yielding a common product The rate constant for the faster reactions ranged from 1.5 to 2.5 min 1 The slower reaction exhibited a greater variability (0.07 to O.23 min −1 ) In the absence of phytate the rate curves were not biphasic and could be fitted to a simple first-order rate equation At metal ion phytate mol ratios of ≦ 2, the rate curves were no longer biphasic and were first order for both the time dependent changes in pH and “absorbance” Furthermore, above a pH of ca. 8 the insoluble phytate complexes formed with either Ca(II) or Zn(II) became soluble This resolubilization phenomenon was not seen with Cu(II):phytate complexes

Thermal titrimetric evaluation of the heats of ionization of the commonly occurring amino acids and their derivatives.

Abstract By using instrumentation of relatively simple design it has been shown that calorimetric data of good precision can be obtained with rapidity and ease of operation. The heats of ionization of all the commonly occurring amino acids have been determined and, depending on the differences in ionization constants, heats of ionization of individual groups in a mixture can also be obtained.

Heat of complex formation of Al(III) and Cd(II) with phytic acid. IX.

The interactions of Cd(II) and A1(III) with phytic acid to form both soluble and insoluble complexes have been studied by calorimetry. The reactions were examined at metal ion: phytate mol ratios ranging from 1-6 in the case of Cd(II) and 1 and 4 in the instance of Al(III). The heats of reactions were endothermic within these mol ratios. The enthalpies of soluble complex formation of Cd(II) are compared to those of other divalent metal cations that were studied under similar conditions. Based on the enthalpies, it is suggested that the order of combining affinity of these metal cations for phytic acid is Cu(II) greater than or equal to Zn(II) greater than or less than Cd(II) greater than Mn(II) greater than Mg(II) greater than Co(II) greater than Ni(II) The heats of precipitations (which includes binding, solvation changes, etc.) to form the insoluble Cd(II)- and Al(III)-phytate complexes were determined. The reactions were endothermic and had enthalpies of 20.2 and 28.7 kcal mol-1, respectively. From measurements of the Cd(II) and Al(III) concentrations, it was calculated that 5.4 mol Cd(II) and 4 mol Al(III) bind per mol of precipitated phytate. Al(III) only formed soluble complexes with phytic acid at mol ratios of metal cation:phytate of 1. This reaction was markedly slower, at least as judged by the thermal decay curves, and had an enthalpy considerably larger in magnitude (6-17-fold) than those of all of the divalent cation-phytate reactions thus far investigated in this manner.

Heats of precipitation of calcium phytate

Abstract The heat of precipitation has been determined for the reaction, in aqueous solution, of calcium ion with the phytate anion. The reaction is endothermic. From the value of the equilibrium constant associated with the reaction, the entropy change has been calculated. The large, positive entropy change is consistent with the view that when calcium ion reacts with the phytate anion there is a decrease in the hydrophilic character of the phytate anion. The quantitative aspects of the interaction of phytic acid with calcium provides a rational basis for the understanding of the mechanism of metal deprivation by phytic acid.

Modification of the amino group of isoleucine-16 in chymotrypsin with retention of activity

Abstract The N-terminal isoleucine-16 residue of δ-chymotrypsin has been modified to the corresponding amidine by reaction with either ethyl acetimidate or methyl picolinimidate. The modified δ-enzymes show no change in specific activity or in the active site concentration but have one less isoleucine amino group as determined by both end-group analysis and potentiometric titration. Despite the fact that the amidinated amino group has a pK′ about 12, k cat K′ m versus pH profiles (acetyl-L-tryptophan ethyl ester as substrate) are bell-shaped with the upper pK′ 9.3. Thus, the Ile-16 amino group is neither essential for chymotrypsin activity nor responsible for the kinetically seen group with pK′ 9.

The interactions of inositol hexaphosphate with Fe(III) and Cr(III). A calorimetric investigation. XV

Abstract The complexation of Fe(III) and Cr(III) with inositol hexaphosphate (phytic acid) has been studied by calorimetry. The reactions were examined at metal ion: phytate mol ratios 1 and 4, in the instance of Fe(III), and 4 only in the case of Cr(III), The heats of reactions were endothermic within these mol ratios. Precipitation occurred in each instance of the Fe(III)-phytate reactions. From measurements of the remaining Fe(III) and phytic acid concentrations, it was calculated that 4.1 mol Fe(III) bind per mol precipitated phytate at initial mol ratio 4:1. In a similar manner it was found at the initial, lower mol ratio of 1:1 that 3.5 Fe(III) bind per mol precipitated phytate. The heats of precipitations (which include binding, solvation changes, etc.) of the Fe(III)-phytate reactions were approximately 16 kcal mol −1 . This value is substantially smaller than those obtained for most previously investigated metal ion-phytate precipitation reactions. Possible reasons for the lower heat of precipitation of the Fe(III)-phytate reaction are discussed. The Cr(III)-phytate reactions were investigated under conditions in which both soluble and insoluble complexes formed. Certain complications, which are detailed, existed in these studies. An unusually large value, i.e., 81 kcal mol −1 , was obtained for the heat of precipitation of the insoluble Cr(III)-phytate complex. Due to the above mentioned complications, this enthalpy of precipitation, together with the experimentally determined value of 11.5 mol Cr(III) per mol phytic acid precipitated, are subject to considerable uncertainty. On the basis of the present, and previously reported results (reference 12), it is proposed that Al(III) has a greater affinity for phytic acid than does Fe(III), which in turn exhibits a greater affinity than does Cr(III).

The heat of complex formation of copper(II) with phytic acid

Abstract The heats of complex formation of Cu(II) with phytic acid to form soluble complexes in the absence of precipitation at acid pH have been measured. The reaction was examined over a wide range of mol ratios of Cu(II):phytate. In all cases the heats of reaction were endothermic. Measurements of the uncombined copper by use of a copper electrode allowed calculation of the combined copper and hence the enthalpies. These latter values varied to some extent, depending upon the Cu(II):phytate ratio and the pH region where the reaction was examined. Factors which could contribute to the variation in the enthalpy terms include changes in the heats of ionization and possible structural changes with Cu(II) bound.

Phytic acid-manganese(II) ion interactions: A calorimetric and EPR study

Abstract The interactions of Mn(II) with phytic acid to form both soluble and insoluble complexes has been studied by calorimetric and EPR (electron paramagnetic resonance) techniques. The reaction to form soluble complexes (in the absence of precipitation) was examined over a range of mol ratios of Mn(II):phytic acid, from 1 to 6. Over this range of mol ratios the heats of reactions were endothermic. The amount of Mn(II) combined with the phytate was measured indirectly by an EPR procedure and the experimental heats of enthalpies were calculated. These enthalpies are a composite of a number of factors and varied to some extent dependent upon the Mn(II):phytate ratio. It is suggested that variations in the enthalpies could be a reflection of changes in the heats of ionization of the phytic acid and/or different binding sites with increasing Mn(II):phytate ratio. The heat of precipitation (which includes binding, solvation changes, etc.) of the insoluble Mn(II)-phytate complex was endothermic (22.4 kcal mol-1). This is similar in magnitude to the reported heats of precipitation reactions of Zn(II), Ca(II), Cu(II), Co(II), and Ni(II) with phytate. At approximately pH 6, measurements of the free Mn(II) concentration suggested that 5 mol Mn(II) binds per mol phytate.

Evaluation of the Electrostatic Interaction Parameter on a Simulated Protein Titration Curve

Abstract Values for the electrostatic interaction parameter, ω, derived from the potentiometric titration curves of proteins may be erroneous unless both the number of groups ionizing with the same ionization constants are known and the titration curve is obtained with great precision and accuracy. The nature of the errors which can occur in the evaluation of u is demonstrated for calculated curves constructed without electrostatic interaction assumed. Since in a protein, the number of groups (and their pKs) titrating in a given pH region is unknown and the accuracy of protein titrations is limited, we suggest that the titration of a protein can be adequately described as the sum of a series of mass-law expressions.

Interactions of inositol hexaphosphate with Pb(II) and Be(II). A calorimetric study. XVIII.

Abstract The interactions of Pb(II) and Be(II) with inositol hexaphosphate (phytic acid) were studied by calorimetry. The reactions were examined at initial pH values of 3.56 and 2.40 with a metal ion:phytic acid mol ratio of six in each instance. Insoluble metal ion-phytate complexes were formed with endothermic heats of precipitation. Analyses by internally coupled plasma (ICP) procedures indicated that 5.3 mols Pb(II) combined per mol phytic acid precipitated at initial pH 3.56, whereas 4.7 mols Pb(II) combined per mol phytic acid precipitated at initial pH 2.40. The corresponding results for Be(II) were 5.0 mols Be(II) combined per mol phytic acid precipitated at initial pH 3.56 and 4.6 mols Be(II) combined per mol phytic acid precipitated at initial pH 2.40. The enthalpy of precipitation of the Pb(II)-phytate reaction was 10.54 kcal at initial pH 3.56 and 6.27 kcal at initial pH 2.40. Similarly, those for the Be(II)-phytate precipitations were 26.79 kcal at initial pH 3.56 and 23.18 kcal at initial pH 2.40. We conclude that, among the numerous divalent metal cation-phytic acid reactions we have previously studied, Pb(II) and Be(II) exhibit the strongest combining affinity for phytic acid.