Interactions of biomolecules with peptide affinity supports

A novel combinatorial peptide library radiological screening method has been developed to identify ligands that bind proteins or small molecules with high affinity and specificity. That any molecule incorporated with 14C is a suitable target using this approach is particularly advantageous for small molecules, where antibody-related detection is impractical and modification of the small molecule is undesirable. The radiological method is highly sensitive: detection at 70 dpm/bead, equivalent to protein levels in the femtomolar range, has been achieved. Furthermore, the method offers direct detection with few parameters, thereby facilitating optimization of blocking and binding conditions. Peptides screened using this technique were shown to purify fibrinogen from mixtures with human serum albumin. The suitability of peptide affinity supports derived from combinatorial peptide libraries for large scale protein purification was illustrated with a resin substituted with the peptide FLLVPL. Active fibrinogen was reproducibly purified from human plasma.;Fibrinogen was found to interact with the FLLVPL surface through both hydrophobic and ionic interactions. The affinity of the protein for the peptide in solution is lower than the affinity of the protein for the peptide when immobilized. Furthermore, the affinity constant increases and the desorption rate constant decreases as the peptide density is increased. This suggests binding cooperativity, either through multi-point interactions or conformational changes in the peptide or protein as the peptide substitution is changed.;At FLLVPL peptide densities below 14 g/l, Tentacle-type resins show greater dynamic binding capacity for fibrinogen than do conventional-type chromatography resins. Modeling suggests fibrinogen binds faster and with greater affinity to the Tentacle supports at the lower peptide densities. The greater flexibility of the peptides on the Tentacle supports may allow them to adopt a configuration that is optimal for binding the target protein.;It was also shown that peptide presentation on chromatography supports influences small molecule enantiomer binding and resolution. Tryptophan enantiomers were separated on a C18 column saturated with LLL peptide. Bonded LLL peptide, as well as bonded LLL-like peptides containing ion-pairing sites, did not separate the enantiomers. Specific affinity ligands were not identified when screening the available peptide libraries against small, chiral molecules.