Effects of Lyophilization on Metabolic Integrity of Red Blood Cells.

Abstract

Abstract : We have used infrared spectroscopy to evaluate the secondary structure of hemoglobin in red cells lyophilized by Dr. Spargo's most recent protocol. As noted for cells lyophilized by earlier methods, the hemoglobin is unfolded in the dried solid, but refolds during rehydration. Many studies with purified hemoglobin (including some of our preliminary work) have indicated that the rate of formation of methemoglobin is greatly increased if the protein is unfolded. If the same relationship is true for hemoglobin in the dried red cells, then generation of methemoglobin will most likely continue as the cells are stored in the dried solid. It may be possible to minimize this problem by developing methods to inhibit lyophilization-induced unfolding more effectively. We are investigating this approach with purified proteins. Dr. Spargo is attempting to improve stabilization of hemoglobin in the red cells by investigating methods to load cells with stabilizers that we have found to be more effective for purified proteins (e.g., trehalose). As another approach to help mitigate this problem, we will soon begin studying means by which to maximize the activity of methemoglobin reductase in rehydrated cells. If we can take advantage of this enzyme system in the red cells, then it may be possible to reverse oxidative damage to hemoglobin that occurs during lyophilization and storage.