Column selectivity in reversed-phase liquid chromatography I. A general quantitative relationship.

Retention factors k have been measured for 67 neutral, acidic and basic solutes of highly diverse molecular structure (size, shape, polarity, hydrogen bonding, pKa, etc.) on 10 different C18 columns (other conditions constant). These data have been combined with k values from a previous study (86 solutes, five different C8 and C18 columns) to develop a six-term equation for the correlation of retention as a function of solute and column. Values of k can be correlated with an accuracy of +/- 1-2% (1 standard deviation). This suggests that all significant contributions to column selectivity have been identified (and can be measured) for individual alkyl-silica columns which do not have an embedded polar group. That is, columns of the latter kind can be quantitatively characterized in terms of selectivity for use in the separation of any sample.

Computer simulation for the convenient optimization of isocratic reversed-phase liquid chromatographic separations by varying temperature and mobile phase strength.

Software is described which allows the rapid development of separations by means of isocratic reversed-phase liquid chromatography (RP-LC), based on the optimization of column temperature (T) and mobile phase strength (%B). For a given sample, four initial experiments are carried out at two different temperatures, using either isocratic or (better) gradient elution. If isocratic experiments are chosen for computer simulation, it is necessary to select appropriate values of %B for these initial runs. Literature data for solute retention as a function of T are reviewed, as a basis for estimating suitable values of %B at the two values of T selected. The use of optimized values of T and %B led to acceptable separations for three representative samples. The prediction of isocratic separation on the basis of initial gradient experiments is more convenient than the use of initial isocratic experiments, but less reliable. When gradient experiments are used, one additional isocratic experiment can improve the accuracy of such predictions by a "reflection" procedure. The latter approach was confirmed for predictions of both isocratic and gradient separation from initial gradient experiments.

Control of column temperature in reversed-phase liquid chromatography.

When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems which can result in a failure of method transfer for non-ambient RP-LC methods were examined. Means for correcting for such effects and thereby ensuring method transferability are described.

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time III. Improving the accuracy of computer simulation.

Previous studies have shown that four experimental runs, where both temperature T and gradient time tG are varied, can be used for the reliable prediction of separation as a function of these two variables (two-dimensional optimization). Computer simulation (e.g., DryLab) can then be used to predict "optimized" conditions for maximum sample resolution using either isocratic or gradient elution. Samples that contain a large number of components (e.g., n>15-20) present a greater challenge. Resolution for these more complex samples is often quite sensitive to small changes in T or tG in turn requiring greater accuracy in predictions that result from computer simulation. In the present study of several samples, we have examined computer simulation errors that can arise from inexact expressions for retention time as a function of T, tG or isocratic %B. Resulting conclusions are applicable to both complex and simpler samples, in either one- or two-dimensional optimization. Means to anticipate and minimize the impact of these predictive errors are examined.

Bactericidal properties of hydrogen peroxide and copper or iron-containing complex ions in relation to leukocyte function.

Various combinations of hydrogen peroxide, reductant (ascorbic acid and superoxide ion), and copper or iron salts and their coordination complexes were examined to determine their cytotoxicity toward several bacteria with diverse metabolic capabilities and cell envelope structures. Four sets of bactericidal conditions were identified, comprising: (1) high concentration levels (5-100 mM) of H2O2 in the absence of exogenous metal ions and reductant; (2) ferrous or ferric coordination complexes plus enzymatically generated O2.- and H2O2 at relatively low steady-state concentration levels; (3) cupric ion plus low concentration levels of H2O2 (1 microM-1 mM) and ascorbate (10 microM-4 mM); (4) cuprous ion (or cupric ion plus ascorbate) in the absence of O2 and H2O2. Rates of losses in viabilities increased proportionately with increases in the concentration of H2O2 in metal-free environments and with each of the components in the Cu2+/ascorbate/H2O2 bactericidal assay system. Oxidant levels required for equivalent killing increased with increasing cell densities of the bacterial suspensions over the range investigated (2 x 10(7)-2 x 10(9) cfu/ml). Other experimental conditions or other combinations of reagents, most notably Fe3+/ascorbate/H2O2 systems, did not generate bactericidal environments. The patterns of response of the three organisms tested, Streptococcus lactis, Escherichia coli, and Pseudomonas aeruginosa, were similar, suggesting common bactericidal mechanisms. However, preliminary evidence suggests that the lethal lesions caused by the various bactericidal conditions are distinct: As discussed, each of the four bactericidal conditions could conceivably be attained within the phagosomes of leukocytes, although none has as yet been identified.

Bactericidal potency of hydroxyl radical in physiological environments.

Rates of radiolytic inactivation of bacteria suspended in N2O-saturated solutions were dramatically increased over normal background levels when the media contained chloride or bicarbonate ions. The bacteria could be protected from this enhanced toxicity by the addition of free radical scavengers (ethanol, ascorbate, hydrogen peroxide, mannitol, glucose, EDTA, picolinic acid), indicating that the lethal reactions were extracellular in origin. Prior irradiation of chloride-containing solutions led to formation of hypochlorous acid, which was identified by detection of ring-chlorinated products when reacted with fluorescein. Prolonged irradiation of other solutions did not lead to accumulation of bactericidal agents; however, irradiation of bicarbonate-containing solutions in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) led to formation of the EPR-detectable DMPO.CO3- adduct. The results are interpreted in terms of formation of secondary radicals, among which the carbonate and chlorine radicals are uniquely toxic to bacteria. From rate comparisons of the solution components, it was concluded that the reactions involving chloride ion are unlikely to be expressed in biological environments, but that the CO3- radical could be an important intermediary oxidant in peroxide-inflicted cellular damage, particularly in spatially confined environments such as the leukocyte phagosome.

Oxygen exchange in the gamma-phosphoryl group of protein-bound ATP during Mg2+-dependent adenosine triphosphatase activity of myosin.

When ATP binds to myosin in the presence of Mg2+ there follows a rapid cleavage reaction to yield a myosin-product complex whose breakdown is rate-limiting in the overall adenosine triphosphatase reaction at 21 degrees and pH 8.0. Recent kinetic studies on this system have led to the proposal that the cleavage of ATP bound to myosin is reversible. This conclusion is based in part on the observation that when ATP is mixed with an excess of myosin active sites a small amount of tightly bound ATP exists whose life-time coincides with that of the myosin-product complex and implies these two species are in equilibrium during their decay. Previous oxygen exchange studies have shown that phosphate released as free product contains more than one oxygen atom from water. A rapid equilibration between myosin-bound ATP and a myosin-products complex can account for the extra water oxygen incorporation of the product phosphate. Such a model requires that the gamma-phosphoryl group of the bound ATP also exchanges its oxygen atoms with water. Results presented in this paper show that protein-bound ATP labeled in the three terminal oxygen atoms of the gamma-phosphoryl group with 18O exchanges about 75% of its label within 2 s of binding to the active site of myosin. This result provides chemical evidence for a model in which bound ATP undergoes a reversible reaction with water. Incomplete exchange may arise from kinetic and/or structural restraints on the mechanism and plausible models are discussed.

Coupling of "high-energy" phosphate bonds to energy transductions.

Recent results suggest consideration of a new concept for oxidative phosphorylation in which a prime function of energy is to bring about release of ATP formed at the catalytic site by reversal of hydrolysis. Data with submitochondrial particles include properties of an uncoupler insensitive Pi=HOH exchange, a rapid reversible formation of bound ATP in presence of uncouplers, and predictable patterns of 32-Pi incorporation into ATP in rapid mixing experiments. ADP is confirmed as the primary Pi acceptor in mitochondrial ATP synthesis, but with chloroplasts ADP is also rapidly labeled. Other findings with pyrophosphatase and with transport ATPase harmonize with the new concept. Measurements of the reversal of ATP cleavage and binding by myosin suggest that oxygen exchanges result from reversible cleavage of ATP to ADP and Pi at the catalytic site and that the principal free energy change in ATP cleavage occurs in ATP binding. Reversal of conformational changes accompanying ATP binding and cleavage is proposed to drive the actin filament in contraction. Thus energy transductions linked to ATP in both mitochondria and muscle may occur primarily through protein conformational change.

Isotopic probes of catalytic steps of myosin adenosine triphosphatase.

A new approach to the direct estimation of the value of the off constant for dissociation of ATP from myosin subfragment 1 (S1) has been developed. From measurements of the extremely slow rate of release of [32P] - ATP formed from 32P(i) by S1 catalysis and the amount of rapidly formed [32P] - ATP tightly bound to S1, the value of the off constant is approximately 2.8 X 10(-4) sec -1 at pH 7.4. The concentration dependencies for P(i) in equilibrium H18 OH exchange and for (32)P(j) incorporation into myosin-bound ATP give direct measurements of the dissociation constant of P(i) from S1. Both approaches show that the enzyme has a very low affinity for P(i), with an apparent K(d) of greater than 400 mM. Measurement of the average number of water oxygens incorporated into P(i) released from ATP by S1-catalyzed hydrolysis in the presence of Mg2+ suggests that the hydrolytic step reverses an average of at least 5.5 times for each ATP cleaved. With the Ca2+ -activated hydrolysis, less than one oxygen from water appears in each P(i) released. This finding is indicative of a possible isotope effect in the attack of water on the terminal phosphoryl group of ATP.

The use of nuclear magnetic resonance to describe relative modes of binding to lysozyme of homologous inhibitors and related substrates.

Proton magnetic resonance has been used to study the association of inhibitors and substrates with hen egg-white lysozyme. Changes in chemical shift, due to association, of acetamido methyl group resonances of the small molecules have been quantitated. This has allowed definition of magnetic parameters for three contiguous binding subsites on the enzyme surface. The relative modes of occupancy of these sites by N-acetyl-D-glucosamine (NAG), chitobiose, chitotriose, their methyl glycosides, and chitotetraose have been delineated. In addition, the binding to these sites of N-acetyl-D-muramic acid (NAM) and a cell-wall disaccharide, NAG-NAM, have been studied. There is good, although not complete, agreement between the results obtained and X-ray analysis studies of the binding of some of these inhibitors to crystalline lysozyme. Binding of synthetic substracts, such as p-nitrophenyl-2-acetamido-4-O-(2-acetamido-2-deoxy-beta-D -glucopyranosyl)-beta-D-glucopyranoside (NAG-Gluc-varphiNO(2)), has also been studied by the magnetic resonance technique described.