(376a) Immobilization of Myoglobin from Horse Skeletal Muscle and Hemoglobin I from Lucina Pectinata in Hydrophilic Polymer Networks for Hydrogen Sulfide Biosensor Application

Lucina pectinata lives in sulfide-rich mud in the coastal mangroves of the southwest coast of Puerto Rico. The bivalve mollusc harbours sulfide-oxidizing chemoautotrophic bacteria and expresses a monomeric hemoglobin I, HbI, with normal oxygen, but extraordinarily high sulfide affinity. Hydrogen sulfide is a highly toxic gas. Hydrogen sulfide reacts with enzymes in the bloodstream and inhibits cellular respiration resulting in pulmonary paralysis, sudden collapse and death. This project proposes to investigate the use of Hemoglobin I from Lucina pectinata as a recognition element that will allow the detection of hydrogen sulfide in a biosensor application. Since HbI have the ability to change its absorbance from 407 nm to 426 nm, it can be used to create a biosensor capable to recognize the presence of hydrogen sulfide. The biosensors are created by the immobilization of HbI by adsorption and entrapment techniques. Hydrogels were selected as immobilization support, which are chemically or physically cross-linked hydrophilic polymer networks that are environmentally sensitive and capable of absorbing large amounts of water. Three different polymer morphologies were investigated and evaluated in their capacity of better immobilization by adsorption or entrapment techniques. Anionic hydrogels composed of methacrylic acid (MAA), cationic hydrogels composed of dimethylaminoethyl methacrylate (DMAEM), and the neutral hydrogel poly(ethylene glycol) monomethyl (PEGMA) ether monomethacrylate (n=200, n=400, n=1000), all crosslinked with poly(ethylene glycol) dimethacrylate (n=200, n=600, n=1000), were synthesized by free radical solution polymerization. Since HbI from Lucina pectinata is not commercially available and must be isolated and purified, myoglobin from horse skeletal muscle was employed to develop all the necessary experimental protocols and to compare the results obtained from HbI. The hydrogels morphologies were characterized by means of the determination of the polymer volume fraction in the swollen state, the number average molecular weight between crosslinks and the correlation length. The partition coefficient was determined as a measure of the affinity of the protein to be immobilized by absorption in the polymer membrane or to stay free in solution using UV spectroscopy concentration measurements. Results indicated that anionic hydrogels tend to have more affinity towards the protein. Release studies were conducted to evaluate the amount of protein permanently immobilized inside the hydrogels and to evaluate the diffusion mechanism that took place during the molecule release and to quantify the diffusion coefficients.