Isolation And Identification Of Human Hemoglobin Fragments & Studying The Effect Of Hemoglobin On Rat E.Coli Vaginal Infection

The aim of this paper was to isolate human hemoglobin and its fragments, detect their antibacterial activity in vitro and investigate the effect of hemoglobin on rat E.coli vaginal infection. The paper was divided into two parts:Partâ… : Isolation, purification of human hemoglobin fragments and comparison of their antibacterial activity in vitro. Objective: To isolate and puritify human hemoglobin fragrments and compare their antibacterial activity in vitro. Methods: Human hemoglobin was isolated from peripheral blood of healthy volunteers or outdated blood. The cellular membranes of red blood cells(RBC) were disrupted by mixing RBC with CH₂Cl₂, hemoglobin was purified by differential centrifugation; The a andβchains of hemoglobin were separated by cation exchange chromatography and gel chromatography; Theαandβchains were cleaved by cyanogens bromide(CNBr), respectively. The cleaved fragments were purified by reverse phase high performance liquid chromatography (RP-HPLC). The molecular characteristics, such as purity and molecular weight, of the aim molecules in each isolation stage were monitored by tris-tricine sodium dodecyl sulphate polyacrylamide electrophoresis(Tricine SDS-PAGE); The antibacterial activity of hemoglobin and its fragments was determined by agrose radial diffusion assay. Results: Using the chromatography, theαandβchains were obtained from the hemoglobin. Two fragments ofα1-32 (about 3KD) andα77～141 (about 7 KD) from a chain, and one fragment ofβ56-146 (about 10KD) fromβchain were prepared by RP-HPLC. Exceptα1-32, all of the hemoglobin,αchain,βchain, a77～141,β56～146 were very potent against Gram-negative bacteria E. coli ATCC25922 and clinical isolated strain E.coli 54080; but no effective on Gram-negative bacteria P. aeruginosa ATCC 27853, clinical isolated strain P. aeruginosa PAO1, Gram-postive bacteria S. aureus ATCC25923 and fungi C. albicans ATCC 10231. Conclusion: The hemoglobin,α/βchain and their fragments had antibacterial activity, but they were mainly active against Gram-negative bacteria E. coli. Except al-32 comparatively lower, the antibacterial activity of hemoglobin,α/βchain and their fragments were similar.Partâ…¡: The effect of human hemoglobin on E. coli vaginal infection in rats. Objective: To detect the effect of human hemoglobin on E. coli vaginal infection. Methods: Rats' bilateral ovaries were resected by operation. Rats were subcutaneously injected with estradiol benzoate to maintain estrus, and hydrocortisone sodium succinate to induce low systemic immunity. After vaginal flushed with PBS repeatedly, the rats were inoculated intravaginally with E.coli clinical isolated strain.The rats were randomized into the experimental group and the control group. The experimental group was administered intravaginally with hemoglobin. The histologically pathological section and the organ index of uterine and vagina was observed, and then the indicator changes of each group before and after treatment were analyzed. Results: With statistical analysis of the organ index of rat uterine cervix and vagina, it showed no difference between the hemoglobin group (the experimental group) and the blank control group (P＞0.05). The comparison between the matrix control group (no treatment after infection) with the former two groups, there was significant difference (P＜0.05). The organ index of the matrix control group increased significant. The degree of inflammation relief in the hemoglobin group was 57%, and the comparison with the matrix control group had statistically significant difference. Conclusion: Human hemoglobin might relieve the inflammation of E. coli vaginal infection in rats.