Interplay of hydrophobic and electrostatic interactions in biopolymer chromatography. Effect of salts on the retention of proteins.

The effect of salt on the retention behavior of proteins in electrostatic and hydrophobic interaction chromatography is described by a three-parameter equation, log k' = A - B log ms + Cms, where k' is the retention factor and ms is the molality of the salt in the eluent. Parameter B, termed the electrostatic interaction parameter, depends on the characteristic charge of the protein and the salt counterion and governs the change of retention with the salt concentration in ion-exchange chromatography. According to the model the magnitude of the hydrophobic interaction parameter C is determined by the hydrophobic contact area upon protein binding at the stationary phase surface and the properties of the salt as measured by its molal surface tension increment. Retention data measured at different salt concentrations in the eluent on a variety of ion exchangers can be fitted to the above equation which yields U-shaped plots of log k' against log ms. The limiting slopes of the appropriate plots at sufficiently low and high salt concentrations can be used to evaluate the electrostatic and hydrophobic interaction parameters, respectively. The approach, which is based on a combination of established treatments of electrostatic and hydrophobic interactions offers a convenient framework for analyzing retention data in biopolymer high-performance liquid chromatography and for the characterization of stationary phases. Furthermore, it may facilitate some characterization of protein molecules on the basis of their retention behavior as a function of the concentration and nature of the salt in the eluent. In the treatment of electrostatic interactions use is made of the counterion condensation theory that is believed to make possible a more comprehensive analysis than the traditional stoichiometric ion-exchange model which assumes binding of the proteins by coulombic interactions at discrete sites. The treatment of hydrophobic interactions is based on an adaptation of the solvophobic theory which predicts that the hydrophobic portion of the free energy of binding is proportional to the hydrophobic contact area and the microthermodynamic surface tension of the aqueous salt solution. Despite its simplicity the theory was successful in explaining the observed effect of the nature and concentration of salt in the eluent, the pH and the effect of the density of fixed charges at the surface of the stationary phase in the absence of specific salt effects.

Regulation of heat production in the inflorescences of an Arum lily by endogenous salicylic acid.

We have recently purified calorigen, the natural trigger for heat production in the inflorescences of Sauromatum guttatum Schott (voodoo lily), a thermogenic plant, and identified it as salicylic acid. Since then an analytical assay was developed that allows the quantitation of salicylic acid in plant tissues. This assay was used to demonstrate that on the day preceding the day of blooming the levels of salicylic acid in the thermogenic organs (appendix and lower spadix) of the voodoo lily increased almost 100-fold, reaching a level of 1 mug/g of fresh weight. The level of salicylic acid in the appendix started to rise in the afternoon and reached its maximum in the late evening, whereas the maximum accumulation of salicylic acid in the lower spadix occurred late at night. The increase in salicylic acid level in the appendix was followed the next morning by a spectacular metabolic burst that lasted for about 7 hr and at its peak increased the appendix temperature by over 12 degrees C. The second, 14-hr-long, thermogenic episode in the lower spadix started late at night and ended on the following morning, after maximum temperature increases of more than 10 degrees C. The concentration of salicylic acid in both thermogenic tissues promptly returned to basal, preblooming levels at the end of the thermogenic periods. The thermogenic response was under strong photoperiodic and developmental control, with salicylic acid eliciting much stronger thermogenic responses in light than in darkness. Similar surges in salicylic acid occurred in nonthermogenic male and female flowers, while the concentration of salicylic acid in the spathe remained consistently below 20 ng/g of fresh weight. Of 33 analogs of salicylic acid tested, only 2,6-dihydroxybenzoic acid and acetylsalicylic acid (aspirin) were thermogenic. The activity of 2,6-dihydroxybenzoic acid exceeded that of salicylic acid.

Phloem Mobility of Xenobiotics: II. Bioassay Testing of the Unified Mathematical Model.

TWO BIOASSAYS WERE USED TO TEST PHLOEM MOBILITY OF SELECTED XENOBIOTIC COMPOUNDS: (a) excised bean leaf assay; (b) rooted bean leaf assay. Compounds assayed were N-alkylpyridiniums with systematic variation in octanol-water partition coefficients (log K(ow)), substituted benzoic acids with about the same log K(ow) value but variable acidities. Results of the assays strongly conform, quantitatively, to the predictions of the unified mathematical model. Results also indicate that the membrane permeability value of a compound, which depends directly on log K(ow) value, is the overriding factor in determining phloem mobility. When the weak acid functionality of a compound confers increased phloem mobility, it does so principally by making the log K(ow) value, and consequently the membrane permeability of the compound more optimal.

Salicylic Acid: a natural inducer of heat production in arum lilies.

For more than 50 years the identity of "calorigen," the agent that triggers pronounced heat production in the flowers and inflorescences of some thermogenic plants, remained obscure. Mass spectroscopic analysis of highly purified calorigen extracted from the male flowers of Sauromatum guttatum Schott (voodoo lily) revealed the presence of 2-hydroxybenzoic (salicylic) acid. Application of salicylic acid at 0.13 microgram per gram (fresh weight) to sections of the upper part of the plant's immature spadix, known as the appendix, led to temperature increases of as much as 12 Celsius degrees. These increases duplicated, in both magnitude and timing, the temperature increases produced by the crude calorigen extract. The sensitivity of appendix tissue to salicylic acid increases daily with the approach of anthesis and is controlled by the photoperiod. Thus, at least in some Arum lilies, salicylic acid functions as an endogenous regulator of heat production.

High-performance liquid chromatography of substituted p-benzoquinones and p-hydroquinones. II. Retention behavior, quantitative structure-retention relationships and octanol-water partition coefficients.

The retention behavior of methoxy-substituted p-benzoquinones and the corresponding hydroquinones in reversed-phase chromatography was examined on octylsilica and two octadecylsilica stationary phases and with five hydroorganic mobile phases containing acetonitrile, methanol or tetrahydrofuran and additionally in most cases (NH3OH)3PO4 used as a reducing and buffering agent. The retention order of benzoquinones and hydroquinones was the same on each stationary phase with either methanol or acetonitrile as the organic modifier. On the other hand, minor differences in the retention order were observed with the various stationary phases. In all cases, satisfactory quantitative structure-retention relationships (QSRRs) were found and the data suggest that the differences in the retention behaviour of octadecylsilicas used in this study are silanophilic interactions which, together with solvophobic interaction contribute to the retention of these eluites. Further analysis showed that QSRRs of sterically crowded molecules must take into account reduced surface area available for binding. The retention data obtained with use of aqueous tetrahydrofuran as mobile phase failed to give rise to satisfactory QSRRs. This was attributed to selective solvation of eluite by tetrahydrofuran and/or nearly equipotent binding of eluite and tetrahydrofuran to stationary phase.

Mobile phase effects in reversed-phase chromatography. VII. Dependence of retention on mobile phase composition and column temperature.

The relationship between logarithmic retention factor, kappa, in reversed-phase chromatography and operating conditions including mobile phase composition and column temperature is still controversial. Earlier, the following analytical relationship was proposed for use with alkyl benzenes: kappa = A1 phi (1 - Tc/T) + A2/T + A3 where phi is volume fraction organic co-solvent of the hydro-organic mobile phase, T is absolute temperature and A1-A3 are parameters appropriate to the eluite and stationary phase. Insofar as its use had not been tested with polar eluites that are retained by only one mechanism, its description of the retention of 54 polar and apolar eluites was examined and compared to equations with other, usually more complex, relationships between retention and operating conditions. The following four-parameter equation described best most data: kappa = A1 phi (1 - Tc/T) + A2/T + A3 + A4 phi. But neither this nor any other four-parameter equation under examination here emerged solely as the best expression for the dependence of retention on the eluent composition and temperature. The average relative errors were 7.8% and 6% with use of the three- and the four-parameter equations which were found to be best for a given eluite, respectively. In view of the small decrease in error but significant increase in complexity and in data points with the use of the four-parameter equation, the three-parameter equation is recommended for general use.

Reversed-phase chromatography of proteins with diphenyl-silica column and hydro-organic eluents containing two organic solvents.

High-performance liquid chromatography of proteins with silica bonded diphenyl stationary phase and hydro-organic mobile phases with linear gradient elution has been carried out with binary organic modifiers. The use of a mixture of 2-propanol and 1-butanol facilitated elution at total organic modifier concentrations significantly lower than with the use of 2-propanol alone. Furthermore higher protein recovery and retention of biological activity was obtained with the binary organic modifier because the increase in eluent strength with binary organic modifier was greater than that of the denaturing strength of the eluent. The use of a short, 3 cm long, column, relatively high flow-rates and steep gradients was also advantageous in attenuating protein denaturation. The results were interpreted within the framework of a theoretical treatment of the combined effect of the retention process and denaturation reaction simultaneously occurring in the column.

High-performance liquid chromatography of substituted p-benzoquinones and p-hydroquinones. I. Interplay of on-column redox reaction and the chromatographic retention process.

Chromatograms exhibiting unusual elution profiles were obtained in the reversed-phase chromatography of methoxy-substituted hydroquinones with hydro-organic mobile phases. The dependence of the elution profiles on the flow-rate and the mass of injected sample indicated that peaks were caused by the interplay of the chromatographic retention process and the concomitant oxidation of hydroquinones in the chromatographic column. The reaction was first investigated in free solution and the first-order rate constants and equilibrium constants were estimated for the oxidation of 2-methoxy-, 2,5-dimethoxy-, 2,6-dimethoxy-, 2,3,5-trimethoxy- and 2,3,5,6-tetramethoxyhydroquinones. The effect of Fe3+ and Cu2+ on the oxidation of 2,6-dimethoxyhydroquinone was also examined and it was found that Fe3+ caused a ten-fold increase in the reaction rate. The oxidation of 2-methoxy- and 2,6-dimethoxyhydroquinones in the chromatographic column showed first-order and zerothorder kinetic behavior at low and high sample loads, respectively. Such change in reaction order is typical for heterogeneous reactions that follow Langmuir-Hinshelwood kinetics and suggests the existence of catalytically active sites on the silica. Upon purging the columns with EDTA, on-column oxidation of the hydroquinones was attenuated so that no reaction zones appeared on the chromatogram. Subsequent loading of the column with Fe3+, however, gave rise to broad reaction zones and possibly to total conversion of the hydroquinones to benzoquinones in the column. In all cases investigated the rate of oxidation was much higher in the column than in free solution. On the basis of the results obtained here, heavy mental-catalyzed reaction at the stationary phase surface is proposed to be the dominant mechanism for the oxidation reaction examined here.

Salt-mediated retention of proteins in hydrophobic-interaction chromatography. Application of solvophobic theory.

Retention behavior in hydrophobic-interaction chromatography was examined within the framework of the solvophobic theory. The principal parameters which determine the effect of salt on the retention are salt molality and the molal surface tension increment of the salt. According to the theory, in the absence of special binding effects, increase in salt molality in the mobile phase or change of salt to one of greater molal surface tension increment will result in increased retention of proteins in hydrophobic chromatography. The theory is expanded to treat retention in gradient elution with linear decrease in salt concentration that is equivalent to linear increase in eluent strength. The results of the simple model lead to an expression with two parameters: the adjusted isocratic retention volume of the eluite with the gradient former and the slope of plot of logarithmic adjusted elution volume against salt molality, lambda. The latter parameter is linearly dependent on molal surface tension increment if no specific interactions between the eluite and the stationary phase and/or salt are present. In practice, deviations are to be expected from the predicted behavior due to such effects. The results of calculations are consistent with experimental results obtained with several proteins as the eluites and various salts in the eluent. Although unique values of the critical parameter lambda could not be obtained from the data, the trends showed that lambda is strongly correlated with the value of the molal surface tension increment. The prediction that increase in salt concentration in the initial eluent leads to increase in retention volume was found to be generally true, even when the isocratic retention volumes obtained with use of eluent having low salt concentration were small. Use of NaClO4 in the starting eluent led in some cases to decrease in retention volume with increase in the salt concentration at the beginning of the gradient elution. This effect may be due to specific binding effects.

Effect of aggregation on the kinetic properties of aspartate aminotransferase.

In this investigation the steady-state kinetic parameters of the alpha subform of aspartate aminotransferase (EC 2.6.1.1) were determined in 0.2 M Tris - HCl, pH 8.0, at 25 degrees C. The kinetic parameters for both the forward and reverse reactions were determined under conditions where the enzyme is monomeric, while only the steady-state parameters associated with the forward reaction could be determined under conditions where the enzyme is dimeric enzyme decreased relative to that of monomeric enzyme, 245 versus 360 s(-1) while the Km for aspartate increased, 3.3 versus 2.6 mM. No significant change in the Michaelis constant for ketoglutarate was observed. The steady-state parameters of dimeric enzyme are slightly altered in 0.1 M Na4 P2O7, pH 8.0, the catalytic center activity and Michaelis constant for ketoglutarate being slightly larger. From the dependence of the initial velocity on enzyme concentration the dissociation constant for the monomer-dimer equilibrium is estimated to be 2 - 10(-8) M. A similar value of the dissociation constant was estimated from Sephadex gel filtration experiments.