RESUMEN
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.