The Studies On Purification, Identification Of Haptoglobin From Cohn Fraction Ⅳ

Haptoglobin (Hp), an acidic glycoprotein component of human plasma a2 globulin, is synthesized mainly in the liver. Haptoglobin functions to bind free plasma hemoglobin and promote its clearance from the circulation, which protect the kidneys from damage by hemoglobin.In 1950s, the researchers began to separate and purify the haptoglobin from the plasma. However, the purification efficiency of these methods is low and the cost is high, which limits large-scale preparation of haptoglobin.Haptoglobin has been licensed in Japan since 1985. However, it is yet to be used clinically in Europe, America and China. There is no main indication, rather a general treatment or prophylaxis in situations of acute hemolysis due to its ability to remove cell-free hemoglobin through the liver and prevent damage to renal tubules.These include burn injuries, trauma resulting from massive transfusion, and prophylactic use prior to a number of different surgical interventions.In this study, the preparation method of Haptoglobin was explored by using the Cohn fraction IV in plasma protein production. The study mainly includes five sections:First, we proved that Hp does exist in the Cohn fraction IV by using SDS-PAGE, immunoblot and mass spectrometry analysis. Non-reducing SDS-PAGE permits clearer visualization of the multimeric composition of haptoglobin and reveals bands approximately at 240 kD, which laid the foundation for the subsequent separation and purification of haptoglobin.Second, this study explored how to purify haptoglobin from two aspects: pretreatment and chromatography.According to the molecular structures and properties of other proteins including albumin, alpha 1 antitrypsin, transferrin in Cohn fraction IV, the fraction was first treated with polyethylene glycol precipitation, ammonium sulfate precipitation and the combination of rivanol and salting out method as pre-treatment prosess respectively. Through comparative analysis, the results showed that the combination of rivanol and salting out method was preferable in the three classical methods. This method can effectively remove the impurity, such as albumin, and target protein enriched, which was benifical to the subsequent chromatography steps. It is a simple, effective pretreatment method for large-scale preparation.In this study, three different chromatography processes DEAE-Sepharose Fast Flow ion-exchange chromatography, QMA Spherosil M ion-exchange chromatography, Q-Sepharose Fast Flow ion-exchange chromatography were used to isolate and purify haptoglobin under different pH conditions, respectively. Through the comprehensive comparison, the use of Q-Sepharose Fast Flow ion exchange chromatography obtained haptoglobin was found to be of good purity and less loss.In addition, the fraction was first treated with the combination of rivanol and salting out method, followed by scale-up of Q-Sepharose Fast Flow ion-exchange chromatography with buffers at pH5.5. Though the recovery of the process was low, the overall process is relatively stable.The activity of haptoglobin obtained by small scale and amplification was 2.8U/mL (10mg/mL),3.75U/mL (15mg/mL) measured by Native-PAGE.Immunoblot analysis and mass spectrometry further proved that the method can be successfully isolated the target protein haptoglobin.The preparation method of haptoglobin was explored by using the Cohn fraction Ⅳ in plasma protein production.We explore the pre-treatment and column chromatography separation of haptoglobin by using the Cohn fraction Ⅳ in plasma protein production. In this study, a two-step protocol for purification of haptoglobin from human plasma fraction IV was designed. The fraction was first treated with rivanol and ammonium sulfate, followed by Q-Sepharose Fast Flow ion exchange chromatography. The purification process was simple and easy to scale-up in production with better prospect. These experimental data might shed some light on further investigations on the building of more simple and affordable purification process of haptoglobin from Cohn IV fraction. In addition, our study was also helpful to improving utilization degree of plasma.