Ethylene glycol diglycidyl ether as a protein cross‐linker: a case study for cross‐linking of hemoglobin

Abstract

AbstractBeing widely applied in the chemical industry, ethylene glycol diglycidyl ether (EGDE) was tried as a protein cross‐linker for cross‐linking of bovine hemoglobin (Hb). The characteristics of the reaction and the products were studied using electrophoresis and multi‐angle laser light scattering coupled with HPLC. At pH 7.5, EGDE could react with the sulfhydryl group and the ε‐amino group, but reacted preferentially with the sulfhydryl group. As the pH increased from 7.5 to 9.5, the reaction took place mainly between the amino groups and EGDE. By controlling the pH and other conditions, intra‐ and inter‐molecularly cross‐linked products with narrow size distribution were obtained. The intramolecular product was about 90% of the reaction mixture. The intermolecularly cross‐linked Hb consisted of 74.7% of dimeric Hb with weight‐average molecular weights (Mw) of 146 kDa, 18.9% of trimeric Hb with Mw of 228 kDa, and 6.4% of oligomer with four to six Hb units, Mw of 375 kDa. The cross‐linking altered the oxygen affinity (P50) and the Hill coefficient of Hb but they are within the range of hemoglobin‐based red cell substitutes. The pI of intramolecularly and intermolecularly cross‐linked Hb increased from 6.7–7.4 to 6.9–7.6 and 8.2–8.8 respectively. The study demonstrated that EGDE could be an alternative to glutaraldehyde for cross‐linking of hemoglobin and other proteins on a large‐scale without significantly impairing the biological activity. Copyright © 2006 Society of Chemical Industry