The Protection of Enzyme Activity for the Preparation of Humanized Polymerized Hemoglobin-Superoxide Dismutase-Catalase-Carbonic Anhydrase.

This work finds suitable enzyme activity protectants to improve the recovery rate of enzyme activity in the preparation of human polymerized hemoglobin-superoxide dismutase-catalase-carbonic anhydrase (PolyHb-SOD-CAT-CA), including trehalose, sucrose, glucose, hydroxypropyl-ß-cyclodextrin, and mannitol.Different types and concentrations of enzyme activity protective agents were added during polymerization to compare their protective ability to enzyme activity and the effect on the properties of hemoglobin. The study found that compared with trehalose, the protective effect of sucrose on CA enzyme activity is non-significant to that on hemoglobin, the recovery rate of SOD, and CAT enzyme activity has significant increased. Glucose, hydroxypropyl-ß-cyclodextrin, and mannitol are unsuitable for the added enzyme activity protective agent of PolyHb-SOD-CAT-CA.The protective effect of sucrose on CA was non-significant with trehalose. The protective effect of sucrose on SOD and CAT enzyme activity was higher than trehalose, and the protective effect reached the maximum when the concentration reached 1.5%.

Preliminary Study on Polymerization between Hemoglobin and Enzymes during the Preparation of PolyHb-SOD-CAT-CA.

The objective of this study was to explore the influence of different factors on the aggregation effect on hemoglobin (Hb) and enzymes during the preparation of Polyhemoglobin-Superoxide dismutase-Catalase-Carbonic anhydrase (PolyHb-SOD-CAT-CA). Several factors including temperatures, pH values, Glutaraldehyde (GDA) amounts and enzymes amounts were investigated systematically to study their effects on the enzymes recoveries and polymerization rates including the Superoxide dismutase (SOD), Catalase (CAT) and Carbonic anhydrase (CA), as well as their effects on the molecular weight distribution of PolyHb-SOD-CAT-CA. Then the oxygen affinity and methemoglobin (MetHb) contents of obtained PolyHb-SOD-CAT-CA were measured to evaluate the effects of enzyme crosslinking on the properties of Polyhemoglobin (PolyHb) moieties in the molecular structure of obtained PolyHb-SOD-CAT-CA conjugate. The results showed that the enzyme recoveries and polymerization rates could be decreased with the temperatures increasing and could be generally kept stable in the physiological pH conditions, but presented only slight changes among the investigated enzyme amounts ranges. Although the GDA concentration increasing could promote the enzyme polymerization rates, the enzyme recoveries decreased in whole. The polymerization rate and molecular size of PolyHb-SOD-CAT-CA conjugate increased with the elevation of temperature and the concentration of GDA. Lastly, the P50 values, Hill coefficients, and MetHb contents of PolyHb-SOD-CAT-CA conjugate with different enzyme crosslinking degrees exhibited no obvious differences with each other. In conclusion, the polymerization reactions between enzymes and Hb molecules could be remarkably affected by temperatures, pH values, and GDA amounts, and the enzyme crosslinking presented no obvious effects on the Hb properties, especially about the oxygen affinity and oxidation degrees.

The Preliminary Study on Preparation Technology of PolyHb-SOD-CATCA - The Effects of Different Extractants.

INTRODUCTION: During the preparation of polyHb-SOD-CAT-CA, the lysate was extracted by toluene. However, due to its serious toxicity and potential application in the production of dangerous explosives, the use of toluene would likely be a restriction of the industrial development of polyHb-SOD-CAT-CA. So, selecting other extraction reagents as alternatives to toluene is necessary to promote the industrialization of polyHb-SOD-CAT-CA. AIMS: The objective of this study is to investigate the application of several organic solvents extraction during polyHb-SOC-CAT-CA preparation process, which include n-haxane and diethyl ether, and also to compare with the existing toluene. METHODS: After extraction with different extractants, the effects of studied organic extractant on the stability of hemoglobin and enzymes include SOD, CAT and CA through monitoring the property indexes include Hb concentration, MetHb content, oxygen affinity of Hb, enzymes activities and so on. RESULTS: The P50 and Hill coefficient of n-hexane group were higher than that in diethyl ether group and toluene group. The MetHb contents, Hb recoveries and enzymes recoveries of n-hexane group and toluene group were much better than that in diethyl ether group. The SOD activity recovery rate in n-hexane experimental group was slightly lower than that in toluene group. However, the CAT and CA recovery rate of n-hexane group was higher than that in toluene group. CONCLUSION: The results of this study suggested that the effects of n-hexane on the properties stability and productivity of polyHb-SOD-CAT-CA were nearly similar with that of toluene, indicating potential reliability and feasibility of n-hexane in the future research and development of polyHb- SOD-CAT-CA.