| The aim of this thesis was to gain a better understanding of how polymers can be used to improve the functional properties of proteins. Proteins are highly sensitive to stimuli such as heat, pH, and other environmental stimuli. Various chemical and physical modifications can be use to improve the structural integrity and functional properties of proteins. This project, a collaborative research effort between the Kasi and Kumar research groups, utilizes polymer material chemistry with biophysical chemistry to explore the synthesis, characterization and functional application of a hemoglobin-polyacrylic acid conjugate, Hb-PAA.;The Hb-PAA conjugates were synthesized by random covalent attachment of the amines on Hb to the carboxylic acids of PAA. The initial synthetic conditions allowed for the formation of a highly soluble conjugate but it was observed that alteration of certain parameters could lead to macroscopic gelation. The Hb-PAA conjugates were characterized by circular dichroism, UV, TEM, and storage stability studies. In order to obtain a conjugate that had optimal high temperature activity and resistance to organic solvents, we next mapped out a small library of Hb-PAA conjugates. By varying Hb/PAA ratio, PAA wt% and EDC/COOH ratio, the conjugates shifted from highly soluble nanogels to macroscopically crosslinked gels.;In addition to improving the biocatalytic properties of Hb, we developed a novel biosensor for detection of small molecules, such as heme. The surface plasmon resonance sensor utilizes the highly specific binding pocket of the heme cofactor to detect micromolar concentrations of heme. The sensing surface was fabricated by covalent immobilization of hemoglobin in a polyacrylic acid matrix in situ, which allowed for a high concentration of protein to be located in the plasmon detection range on the Au chip. Removal of the heme unit from the hemoglobin-polymer conjugate (Hb-PAA) resulted in a surface anchored apoHb-polymer conjugate which showed high affinity and selectivity for heme. This apohemoglobin-polyacrylic acid system demonstrates a new concept in SPR detection with the use of protein cofactor binding pockets for heme detection. |
