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

摘要

Part A: This thesis describes the first detailed study ofhow a catalytic immune response matures over a series ofimmunizations. Specifically, three male New Zealand White rabbitswere immunized with a 4- (carboxy) benzyl phenyl phosphate haptenconjugated to keyhole limpet hemocyanin (KLH) . The three rabbitsreceived an initial immunization with the immunogen, followed bythree immunizations at three week intervals. Serum samples wereobtained 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 eachsample was demonstrated by the rate enhancement and catalyticefficiency of the hydrolysis of the corresponding carbonatesubstrate. The results show a remarkable similarity in the immuneresponse maturation seen in two of the three rabbits. The thirdrabbit was diseased and lagged slightly behind. Through the fourimmunizations, the immune response continued to optimize due to anapparent increase in the affinity of the polyclonal antibodies.This is quite significant, in that it provides the firstexperimental evidence that affinity maturation is important forantibody catalysis. We have thus begun to shed the first rays oflight inside the black box that produces catalytic antibodies. Part B: A novel sapphyrin-functionalized silica gel hasbeen used to study the anionic binding properties of the expandedporphyrin sapphyrin. The preparation of the first generation ofthis type of support is discussed. The silica-bound sapphyrindisplayed longer retention times under neutral, isochratic HPLCconditions, for molecules with sterically accessible oxyanions. Inparticular, the sapphyrin-functionalized silica gel showed aremarkable preference for arsonates and phosphates. Furthermore,mixtures of nucleotides and oligonucleotides were cleanly separatedaccording 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 alsoprovides a viable method to selectively separate and analyze aniontypes. These types of modified silica gels may be useful forsolving present day environmental problems, as well as separatingand analyzing phosphorylated biologically relevant molecules.