Studies On The Isolation And Purification Of Porcine Hemoglobin And Polymerized Porcine Hemoglobin

Isolation and purification of porcine hemoglobin and polymerized porcine hemoglobin was studied in this paper.By comparing DEAE-Sepharose Fast Flow, SOURCE 30Q and Q Sepharose XL on the effects of porcine hemoglobin purification, DEAE-Sepharose Fast Flow chromatographic media was chosen to isolate and purify porcine hemoglobin.On this basis,two experiments processes were designed.In the process A,the porcine hemoglobin was extracted by low permeability method,and then purified by ultrafiltration,DEAE Sepharose Fast Flow column and Sephadex G-75 column.In the process B,the porcine hemoglobin was extracted by heat treatment,and then purified by ultrafiltration and DEAE Sepharose Fast Flow column. By comparing the two routs,process B was chosen to purify procine hemoglobin.The chromatographic condition was as follows:buffer A was 20 mmol/L PB(pH7.2) containing 30 mmol/L NaCl,buffer B was 20 mmol/L PB(pH7.2)containing 1.0 mol/L NaCl,firstly chromatographic column was balanced by buffer A,then sample was introduced,secondly the column was eluted by buffer A,next buffer B after the sample flowed through column completely.The flow rate of mobile phase was 2.5cm/min,and the detected wavelength was 280nm.Through this two-step purification process,the obtained hemoglobin's purity was more than 99.9%detected by SDS-PAGE,HPSEC and HPIEC.The total protein recovery of this purification process was 90.3%,and the protein concentration was 7.46g/dL.The characteristics of the process were fast separation rate,high efficience,simple operation and linearity scale-up.The glutaraldehyde polymerized porcine hemoglobin was prepared by the purified porcine hemoglobin according to the applied petant of our laboratory.The molecular weight range of the polymerized hemoglobin was approximately 65kD～800kD detected by gel filtration column combined with multi-angle laser light scattering instrument.The DEAE Sepharose Fast Flow chromatographic column was used to purify the polymerized hemoglobin.The DEAE Sepharose Fast Flow chromatographic column first eluted with the 20 mmol/L PB(pH7.2)containing 50 mmol/L NaCl for 30 min,and separately followed by the elution with a linear gradient of 50～500 mmol/L NaCl containing 20 mmol/L PB(pH7.2)for 50 min and then with 1.0 mol/L NaCl containing PB(pH7.2)for 20 min.The average molecular weight of the purified polymerized hemoglobin was about 300kD,and the content of four polymerized hemoglobin was less than 5%,which reached the process requriements.