SYNTHESIS, CHARACTERIZATION AND IMMUNOLOGICAL STUDIES OF SIX KNOWN-SEQUENCE POLYMERS CONTAINING TYROSINE, GLUTAMIC ACID, ALANINE, AND GLYCINE

The protected tetrapeptide N-(alpha)-tert-Butoxycarbonyl-(gamma)-benzylglutamyl-O-benzyltyrosylalanylglycine was synthesized using N-hydroxysuccinimide esters of the amino acids. The pentachlorophenyl-activated monomer was made, then deprotected at the (alpha)-position and polymerized in benzene. The synthesis proved to have difficulties in both removing reaction by-products and in polymerization. It is possible that an aggregate may have been used for further characterization. The polymer, poly (L-Tyr-L-Glu-L-Ala-Gly) (TGAgly), was synthesized as previously described. The molecular weight of this preparation of TGAgly was found to be 107,000. Circular dichroic, difference spectroscopic, and viscosity studies indicated that the six known sequence polymers poly (L-Tyr-L-Glu-L-Ala-Gly), poly (L-Tyr-L-Ala-L-Glu-Gly), poly (L-Glu-L-Ala-L-Tyr-Gly), poly (L-Glu-L-Tyr-L-Ala-Gly), poly (L-Ala-L-Glu-L-Tyr-Gly) and poly (L-Ala-L-Tyr-L-Glu-Gly) existed in varying amounts of secondary structure between pH 4.5 and pH 9.0. From these studies it was determined that a structural transition occurred over this pH range in these polymers. However, the amount of conformation found at any given pH varied among the polymers. The polymer TGAgly was found to contain about 11% helix at pH 7.2 and 27% at pH 6.0. Antibodies to TGAgly were raised in C57BL/6 mice. These antibodies contained (gamma)(,1) heavy chains and (kappa) light chains. Approximately 30% of the antibody population required the hydroxyl of tyrosine for binding while 70% did not. From sephacryl column studies, it was found that at pH 7.2, in antibody exces, the most commonly found combining ratio was three antibody molecules per TGAgly molecule. At pH 6.0, the combining ratio was five antibodies per TGAgly molecule. This was consistent with the physico-chemical data showing that the amount of helix approximately doubled over this pH range. This data suggested that the maximum size of the anti-TGAgly antibody combining site was between 27 and 38 (ANGSTROM). A component which binds to TGAgly at pH 6.0 was isolated from normal mouse serum. This component was identified by various immunochemical methods as Gc globulin (Vitamin D Binding Protein). It was also found that this protein binds to other anionic polypeptides and inhibits binding of antibodies to most of these polypeptides at pH 6.0