Biomedical subjects

C T Mant

Publications and source records attributed to C T Mant.

40 records · Page 3Linked to original sources

PubMed1985

Separation of peptides by strong cation-exchange high-performance liquid chromatography.

The effects of pH and gradient conditions on the separation of a series of ten peptides (9-36 residues) and carboxamidomethylated troponin I (CM-TnI, 178 residues) on a new commercially available strong cation-exchange silica based 300-A column (Synchropak S300) were examined. The elution times of the peptides were linear with respect to their net charge at pH 3.0 and pH 6.5. The basic protein CM-TnI (pI approximately 9.5) and peptides with net charges from +2 to +10 were separated with linear AB salt gradients varying from 5 mM to 10 mM B per min (A = 5 mM KH2PO4 buffer, pH 6.5 or 3.0; B = 5 mM KH2PO4 buffer, pH 6.5 or 3.0, containing 1 M KCl). All peptides and CM-TnI were eluted with KCl concentrations below ca. 0.6 M. The advantage of performing cation-exchange chromatography over anion-exchange chromatography was demonstrated for the separation of peptides which, while acidic or weakly basic at neutral pH, through protonation of the acidic functions results in positively charged peptides at pH 3.0.

Animals↗

PubMed1985

General method for the separation of cyanogen bromide digests of proteins by high-performance liquid chromatography. Rabbit skeletal troponin I.

In this study, we have demonstrated the necessity for a combination of size-exclusion, ion-exchange and reversed-phase high-performance liquid-chromatography to resolve completely a protein digest. Our approach minimizes the number of steps, and the column order provides the maximum information about the properties of the fragments. The order is: (1) size-exclusion (Bio-Rad TSK-250 column), (2) strong cation-exchange (Synchropak S300 column), and finally (3) reversed-phase chromatography (Ultrapore C3). It was desirable for the first step of the procedure to be size-exclusion chromatography to produce the least number of fractions. The volatile eluent used in size-exclusion eliminated the need for subsequent sample desalting. Volatile buffers were not necessary for the ion-exchange chromatography, since the fractions were both desalted and purified in the final reversed-phase step. All column effluents were compatible with absorbance measurements at 210 nm to provide maximum sensitivity for peptide detection. The results obtained in this study strongly suggest that the combined use of three methods of separation, which utilize different selectivities (size, charge, hydrophobicity), can provide excellent resolving power for peptide separations. We believe this fast, efficient procedure should be generally applicable to other protein digests.

Animals↗

PubMedDate not supplied

Effect of preferred binding domains on peptide retention behavior in reversed-phase chromatography: amphipathic alpha-helices.

A nonpolar environment, such as the hydrophobic stationary phase of a reversed-phase chromatographic packing, may induce helical structures in potentially helical molecules. If a molecule becomes helical on binding and contains a preferred binding domain, as in the case of an amphipathic helix, then some residues may not be contributing to the same extent to the overall hydrophobicity of the peptide. Amphipathic alpha-helical structures may play an important role in protein folding and the interaction of amphipathic alpha-helices with a hydrophobic surface during RPC is likely to be a good mimic of their hydrophobic interactions with other hydrophobic regions in the folded protein. We have designed and synthesized two sets of model peptides of 7, 14, 21, 28 and 35 residues having the same composition but different sequences (Ac-Lys-Cys-Ala-Glu-Gly-Glu-Leu-[Lys-Leu-Glu-Ala-Gly-Glu-Leu]n-amide and Ac-Lys-Cys-Ala-Glu-Leu-Glu-Gly-[Lys-Leu-Glu-Ala-Leu-Glu-Gly]n-amide, where n = 1-4). Circular dichroism studies demonstrated that both sets of peptides had a high potential to form alpha-helical structure in a nonpolar environment, one set representing amphipathic alpha-helical structures and the other set representing non-amphipathic alpha-helical structures. Size-exclusion chromatography confirmed that all of the peptides in both sets were monomeric when bound to a reversed-phase matrix and also under the conditions used for circular dichroism measurements. Peptides with the same amino acid composition and similar secondary structure could be separated by reversed-phase chromatography. The difference in retention time between peptides of the same length increased with the peptide chain length, ranging from a difference of 2.9 min on a C8 column for the two 14-residue peptides up to a maximum difference of 7.3 min for the 35-residue peptides. From the observed and predicted retention times of these two sets of peptides during reversed-phase chromatography, we have demonstrated that it is possible not only to predict the retention behavior of amphipathic alpha-helices during reversed-phase chromatography, but also to deduce the presence of amphipathic alpha-helical structure in peptides based upon their retention data. If from studies such as these we are eventually able to predict, from only amino acid sequence information, the secondary structure of a peptide on binding to a hydrophobic matrix, we may be able to extrapolate this predictive facility to the conformation of the same sequence in larger polypeptides or proteins.

Amino Acid Sequence↗

PubMedDate not supplied

Protein design using model synthetic peptides.

We have designed and synthesized a small, unique protein molecule with defined secondary, tertiary and quaternary structure. This 35-residue peptide, containing a cysteine residue at its N-terminal end, was oxidized to form a 70-residue disulfide-linked two-stranded alpha-helical coiled-coil with the two alpha-helices parallel and in-register. The major contribution to the formation and stabilization of the alpha-helical coiled-coil is hydrophobic interactions between positions 2 and 5 of the heptapeptide repeat (Lys-Leu-Glu-Ala-Leu-Glu-Gly). The protein (L-protein) contains nine leucine-leucine hydrophobic interactions between the alpha-helices of the coiled-coil. Circular dichroism studies demonstrated that this protein in its reduced ([L (r)] or oxidized (L (o)] state was essentially 100% alpha-helical ([theta]220 = -34,050 and -32,000 degrees respectively) at pH 2 (0.1% aqueous trifluoroacetic acid). Our objective was to modify systematically the structure of L to delineate the contribution that various amino acid side chains make to the formation and stabilization of its three-dimensional structure. A-protein, which contains alanine instead of leucine at positions 16 and 19 of the hydrophobic repeat in each chain of the coiled-coil, was compared to the L-protein. At pH 2, the oxidized form of the A-protein [A (o)] was essentially 100% helical. However, the protein was much less stable to temperature denaturation compared to the L-protein. The replacement of two leucine-leucine interactions by two alanine-alanine interactions has a dramatic effect on the formation and stability of the two-stranded alpha-helical coiled-coil structure. The results of this study clearly demonstrate the validity of this synthetic model protein approach to understanding the molecular aspects responsible for the folding and stabilization of protein molecules.

Amino Acid Sequence↗