(163at) Assessment of the Biological Stability of Hemoglobin I from Lucina Pectinata and Myoglobin from Horse Skeletal Muscle in Ionic Hydrophilic Polymer Networks

Hemoglobin I (HbI) from the clam Lucina pectinata is a unique heme-protein as it binds hydrogen sulfide (H2S) with great affinity in the presence of oxygen. This binding event produces obvious absorbance variations that can be monitored by spectroscopic techniques. Therefore, this project proposes encapsulating HbI in hydrogel membranes in order to create the recognition element for an H2S biosensor. The main objective is to examine the physico-chemical interactions between HbI and hydrogel membranes utilizing the entrapment technique. The hydrogel membranes utilized were anionic methacrylic acid (MAA) and cationic dimethylaminoethyl methacrylate (DMAEM), both crosslinked with poly (ethylene glycol) dimethacrylate (PEGDMA) (n=200, 600, 1000). These were synthesized via free radical solution polymerization by ultraviolet radiation. All experimental protocols were designed utilizing equine skeletal muscle myoglobin (Mb) since it is commercially available, whereas HbI must be obtained from wild clams and purified. Protein stability and activity studies were performed for both proteins. Sodium dithionite (SD) was used to reduce both proteins (remove oxygen). Carbon Monoxide (CO) was utilized to analyze activity after reduction. Changes in absorbance were monitored by UV-spectroscopy. Encapsulation of metaquo Mb in MAA-PEGDMA1000 brought about a hypsochromic shift of 13.5nm from its characteristic peak of 408nm. Subsequent reduction of the protein was verified by an increase in the wavelength of the characteristic peak of deoxy Mb. However, the position of this peak was a hypsochromic shift from the usual wavelength of 435nm. Addition of CO to the hydrogel produced a peak displacement but the peak wavelength did not coincide with the characteristic peak of carboxy Mb at 424nm. Similar behaviors after reduction and oxidation with CO were observed for HbI entrapped in the same hydrogel morphology. Displacement of the characteristic peaks of both Mb and HbI may be due to heme distortions or to unfolding or instability of regions far away from the active center. Further studies are necessary to corroborate these hypotheses. Stability studies of Mb in the DMAEM monomer at pH 9.25 show agglomeration and precipitation of the protein. UV spectrums of Mb in DMAEM neutralized with HCl at pH 7.4, 7.08, 6.14 show a displacement in the Soret band for metaquo Mb from 408nm to 400nm. Other methods of encapsulation such as crystallization will be studied. Release studies will be conducted to determine the amount of protein immobilized within the anionic and cationic hydrogel membranes. Studies with H2S will be conducted in order to determine the formation and dissociation times of the HbI-SH2 complex.