Ashok K. Taneja

Prediction of peptide retention times in reversed-phase high-performance liquid chromatography I. Determination of retention coefficients of amino acid residues of model synthetic peptides

We have examined the contribution of individual amino acid residues to peptide retention on reversed-phase (RP) columns by measuring their effect on retention of a model synthetic peptide: Ac-Gly-X-X-(Leu)3-(Lys)2-amide, where X is substituted by the 20 amino acids found in proteins. Consistently similar resolution of the 20 peptides on several RP columns enabled the determination of empirical sets of retention coefficients, describing the hydrophobicity of amino acid residues at pH 2.0 and pH 7.0. The much superior resolution and selectivity obtained with acetonitrile, compared to 2-propanol and methanol, confirmed its value as the best organic eluent for most practical purposes. The necessity of using peptides rather than alkylphenones as internal standards for peptide retention prediction is demonstrated and underlined by the predictive accuracy of our coefficients when applied to the resolution of a mixture of five commercially available synthetic peptide standards on several RP columns. Rules for retention time prediction for linear elution gradients, employing our hydrophobicity parameters, of peptides of known composition are presented and enable the researcher to correct for: (a) instrument variations, (b) varying length or diameter of RP columns, (c) varying n-alkyl chain length and ligand density of RP packings and (d) column aging.

Effects of high-performance liquid chromatographic solvents and hydrophobic matrices on the secondary and quarternary structure of a model protein : Reversed-phase and size-exclusion high-performance liquid chromatography

Abstract A series of five synthetic peptide polymers of 8, 15, 22, 29 and 36 residues with the sequence Ac-(Lys-Leu-Glu-Gly)n-Lys-amide where n = 1–5 was used to examine protein denaturation during reversed-phase high-performance liquid chromatography (HPLC). Size-exclusion HPLC on a TSK G3000SW column was used to show that the 8-, 15- and 22-residue peptides are monomers in the starting solvent for reversed-phase chromatography, 0.1% aqueous trifluoroacetic acid (TFA) while the 29- and 36-residue peptides are dimers. These dimers have been shown to be extremely stable two-stranded α-helical coiled coils where the subunits are held together by hydrophobic interactions [S. Y. M. Lau, A. K. Taneja and R. S. Hodges, J. Biol. Chem., (1984) in press]. In contrast, all five peptides are monomers in acetonitrile—0.1% aq. TFA (1:1). These five peptides were separated by reversed-phase chromatography with an increasing gradient of 0.7% acetonitrile per minute on three matrices of varying carbon loading, pore size, and alkyl chain length (C3, C8 and C18). In all cases a linear relationship between the natural logarithm of the monomeric molecular weight and retention volume was obtained for these peptides, indicating that the 29- and 36-residue dimers had been dissociated on binding to the reversed-phase columns. These results strongly suggest that the vast majority of proteins are denatured upon binding to the hydrophobic matrix. This series of peptides, 900–8000 molecular weight range, was also used for two additional purposes; firstly, to evaluate the reversed-phase columns and, secondly, to evaluate the relationship of ln molecular weight versus retention volume on the TSK G3000SW column.

Denaturation and the effects of temperature on hydrophobic-interaction and reversed-phase high-performance liquid chromatography of proteins : Bio-gel tsk-phenyl-5-pw column

Cytochrome c, myoglobin and lysozyme, as well as two synthetic peptides, TM-22 and TM-36, were used to examine denaturation of protein structure on a hydrophobic-interaction column, the Bio-Gel TSK-Phenyl -5-PW high-performance liquid chromatography column. The first three proteins were chosen because they have a monomeric structure while both synthetic peptides, which have the sequence Ac-(Lys-Leu-Glu-Ala-Leu-Glu-Gly)inn-Lys-amide where n  3 for TM-22 and n  5 for TM-36, are dimeric under solvent conditions used for hydrophobic-interaction chromatography. Only TM-36 is dimeric under reversed-phase conditions. Thus, denaturation of both tertiary and quaternary structure can be examined. The column was operated in both reversed-phase and hydrophobic-interaction modes. This, in combination with temperature variation between approximately 0-50°C, provided conditions where denaturing effects of the support could be examined. In reversed-phase mode, cytochrome c, myoglobin, and TM-22 were eluted in a denatured form throughout the temperature range. In contrast, lysozyme and TM-36 eluted primarily in their native conformation at low temperatures, but experienced partial or total denaturation at higher temperatures. Significantly, all of the polypeptides studied were denatured at room temperature by a conventional reversed-phase column, the Altex Ultrapore RPSC C-3 indicating that the Bio-Gel TSK-Phenyl-5-PW column is less denaturing. In the hydrophobic-interaction mode, the dimeric structure of TM-22 was totally disrupted at all temperatures, while TM-36, myoglobin, and cytochrome c underwent various degrees of partial denaturation as the temperature increased. Comparison of the temperatures at which the various polypeptides underwent denaturation on the column with their normal melting temperatures (where half the molecules are unfolded) demonstrated that the hydrophobic column itself, rather than the temperature, was primarily responsible for denaturation. Hence, even relatively “gentle” hydrophobic columns can promote denaturation of protein structure. Since the tertiary and quaternary structures of most proteins are stabilized by hydrophobic interactions, the possibility of denaturation must always be taken into consideration when a hydrophobic column is used.

Prediction of peptide retention times in reversed-phase high-performance liquid chromatography II. Correlation of observed and predicted peptide retention times factors and influencing the retention times of peptides

Abstract We have assessed the accuracy of a set of amino acid residue retention coefficients by applying them to the prediction of the retention times of 58 peptides under linear gradient elution conditions (solvent A = 0.1% trifluoroacetic acid in water, and solvent B = 0.1% trifluoroacetic acid in acetonitrile). These coefficients were determined by examining the retention times of synthetic model peptides in reversed-phase chromatography. The high degree of correlation (0.98) between predicted observed retention times not only indicated good predictive accuracy for our coefficients but was also further evidence that composition is generally the major factor affecting peptide retention time. For optimum accuracy in retention time prediction on any single column, it was essential to include an internal peptide standard in each run to correct for run-to-run deviations and column aging. The resolution of five commercially available synthetic peptide standards was found to improve with increasing flow-rate and decreasing gradient steepness. Increasing temperature resulted in a decrease in peptide retention times and slightly improved resolution. Rules for retention time prediction are presented which not only enable the experimenter to correct for instrument and column (length, diameter, n -alkyl chain length and ligand density) specifications, but also allow the prediction of peptide retention times at any gradient steepness, flow-rate and temperature.

Separation of hydrophobic peptide polymers by size-exclusion and reversed-phase high-performance liquid chromatography

Abstract Four synthetic leucine polymers of 13,22,26 and 30 residues with the sequence Ac-(Leu) 10 -(Lys) 2 -Ala-amide and (Lys) 2 -Gly-(Leu) n -(Lys) 2 -Ala-amide, where n = 16, 20 and 24, were used to determine the utility of reversed-phase chromatography in separating extremely hydrophobic peptides. Two mobile phases were examined, water—acetonitrile and water—2-propanol, both containing 0.1% trifluoroacetic acid (TFA). 2-Propanol was the preferred organic solvent, and the hydrophobic peptides (13, 22 and 26 residues) were resolved on the Altex Ultrapore C 3 column (300-A pore size, 5-μm particle size, 2.9% carbon loading) in the 25–50% range of a linear AB gradient (A = 0.1% aq. TFA; B = 0.1% TFA in 2-propanol) increasing at 1% B/min. This procedure permits detection by absorbance at 210 nm, and the peptides can be recovered by evaporation or lyophilization. The 30 residue leucine polymer could not be eluted from the reversed-phase column. These peptides showed non-ideal behavior on size-exclusion chromatography in 0.1% aq. TFA containing 50% acetonitrile. However, this caused no problem in peptide purification. The resolution on a TSK G3000SW column was superior to that obtained on TSK G2000SW.

Contact and dipolar contributions to lanthanide-induced NMR shifts of amino acid and peptide models for calcium binding sites in proteins☆

1H nuclear magnetic resonance has been employed to study the binding of Nα-acetyl-l-aspartic acid and Nα-acetyl-l-aspartyl-l-glycyl-l-aspartylamide to the series of six lanthanide ions Dy3+ through Lu3+. Values for the dissociation constants and the maximum lanthanide-induced shifts were obtained by fitting the titration data for each metal ion to appropriate binding curves. The shifts were separated into contact and dipolar terms without prior knowledge of the symmetry of the complex or the orientation of the principle axis system of the magnetic susceptibility tensor. The results indicate the contact shifts in 1H NMR are not always negligible, and that Yb3+ appears to be the best calcium analog for structural studies when the contact interaction is significant.

Separation of basic peptides by cation-exchange high-performance liquid chromatography

Chromatographic separations of a series of highly basic peptides on commercially available 300—A pore size CM 300 weak cation-exchange columns have been compared at various loads, pHs and ionic strengths of the eluent. On analytical columns (250 x 4.1 mm I.D.), mixtures of basic peptides containing 7 – 9 nmole of each component were separated with a 50 mM KH2PO4–KC1 gradient (pH 4.5) and under isocratic conditions (pH 4.5 and 6.5). The isocratic conditions demonstrated the effects of pH and ionic strength on retention time and resolving power on the CM 300 column. The load capacity of a CM 300 preparative column (250 x 10 mm I.D.), studied under gradient conditions (50 mM KH2PO4, 0.2 – 0.4 M KC1, pH 4.5 and 6.5), revealed that its capacity is much greater at pH 6.5. Loads up to 10–20 mg (6.6–13.3 μmol) could be applied before peaks in the crude peptide sample tested were seen to fuse.

SUBGENOMIC RNA OF ARENAVIRUS PICHINDE AND ITS IMPLICATION IN REGULATION OF VIRAL GENE EXPRESSION IN PRODUCTIVE INFECTION AND PERSISTENCE

Publisher Summary This chapter discusses the role of Pichinde in the regulation of viral gene expression in productive infection and persistence. Search for primary gene products and the assignment of gene loci on the viral RNA of Pichinde virus conveyed a picture of a virus of very simple genetic constituents. It consisted of 2 viral RNA and 3 viral proteins. The limited sequence data available of the S RNA also suggested a very simple arrangement of genes on the RNA. However, Pichinde virus, as well as other arenavirus, was able to exercise a wide range of gene expression observed in the replication of virus and also in persistent infection. The infected cell produced extra cellular virus at a logarithm rate for the first 2 to 3 days of infection. Then the rate of virus production was reduced abruptly for two logs during the following days. This change in extracellular virus titer was likely because of a concomitant 2 log decrease of virus infectious centers in the cell culture. It suggested that more than 95% of the cells in culture had shut off virus production, while the remaining 1 to 5% of the cells still producing virus contributed to the reduced level of virus released into the culture medium.