A: The progression of a catalytic immune response; B: molecular recognition of anions by silica bound sapphyrin

Abstract

Part A: This thesis describes the first detailed study of how a catalytic immune response matures over a series of immunizations. Specifically, three male New Zealand White rabbits were immunized with a 4- (carboxy) benzyl phenyl phosphate hapten conjugated to keyhole limpet hemocyanin (KLH) . The three rabbits received an initial immunization with the immunogen, followed by three immunizations at three week intervals. Serum samples were obtained from each rabbit ten days following every immunization. Pure polyclonal IgG antibodies were obtained by a multiple step isolation and purification process. The catalytic activity of each sample was demonstrated by the rate enhancement and catalytic efficiency of the hydrolysis of the corresponding carbonate substrate. The results show a remarkable similarity in the immune response maturation seen in two of the three rabbits. The third rabbit was diseased and lagged slightly behind. Through the four immunizations, the immune response continued to optimize due to an apparent increase in the affinity of the polyclonal antibodies.This is quite significant, in that it provides the first experimental evidence that affinity maturation is important for antibody catalysis. We have thus begun to shed the first rays of light inside the black box that produces catalytic antibodies. Part B: A novel sapphyrin-functionalized silica gel has been used to study the anionic binding properties of the expanded porphyrin sapphyrin. The preparation of the first generation of this type of support is discussed. The silica-bound sapphyrin displayed longer retention times under neutral, isochratic HPLC conditions, for molecules with sterically accessible oxyanions. In particular, the sapphyrin-functionalized silica gel showed a remarkable preference for arsonates and phosphates. Furthermore, mixtures of nucleotides and oligonucleotides were cleanly separated according to the number of phosphate units they possessed. Thus, not only does sapphyrin-bound silica gel provide an exquisite way to investigate anion binding specificities of sapphyrin, it also provides a viable method to selectively separate and analyze anion types. These types of modified silica gels may be useful for solving present day environmental problems, as well as separating and analyzing phosphorylated biologically relevant molecules.