Preparation Of Heme-β-cyclodextin Inclusion And Its Binding With Proteins

Heme plays its biological roles through three ways: hemoproteins,heme-responsive proteins and heme-dependent reactive oxygen species(ROS). First,the major form of heme in organisms is as the prosthetic group of numerous diverse hemoproteins, such as hemoglobin, myoglobin, cytochromes and peroxidases;Second, free heme is a regulator of numerous diverse heme-responsive proteins which can be protein kinases, transcription factors, signal transduction receptors, ion channels and so on. In the third place, free heme produces ROS through NADPH oxidases and Fenton reaction and ROS involves in a diverse array of signaling pathways by controlling the thiol/disulfide redox states of proteins. In addition, as a dangerous signal, free heme can binds to TLR4, one of the pattern recognition receptors of innate immunity, leading to the activation of innate immune response/inflammation. Furthermore, our laboratory has proved that free heme has the potential to be developed into a safe adjuvant.However, heme is hard to be solved in water on the condition of physiological p H,which is inconvenient to be used as an immune adjuvant. To test immune adjuvant effects of heme, our laboratory made heme-liposome inclusion. But this method is only suitable for basic experimental exploration, because liposome can be broken down easily and be hard to store for a long time. So it is very important to find aformulation for heme adjuvant with good safety, convenient storage and easy use.Beta-cyclodextin can include hydrophobic drug to form drug-β-cyclodextin inclusion to increase the water solubility of the drug and there are no poisonous substances in the preparation process. The drug-β-cyclodextin can be dried into powder for the long time storage and the use by redissolving it. The aim is to study the preparative technology of heme-β-cyclodextin inclusion and the interaction between protein antigens and heme-β-cyclodextin inclusion.An optimized protocol was established for the massive preparation of heme from fresh animal blood. Purity of the prepared heme was more than 98% by the identification of absorption spectra and the yield rate reaches 4.7g per liter of blood.Heme-β-cyclodextrin inclusion was prepared by mixing saturated β-cyclodextrin water solution with heme-ethanol solution under ultrasonication. By designing orthogonal tests, the best conditions for the formation of heme-β-cyclodextrin inclusion were:(1) the ratio of the volume of ethanol to the quality of the heme is10:1;(2) the ratio of the volume of water to the quality of β-cyclodextrin is 50:2;(3)the temperature and time of reaction are35℃ and 80 min, respectively.To study whether the prepared heme-β-cyclodextrin inclusion can be used as a better formulation for immune adjuvant, the combination of the prepared heme-β-cyclodextrin inclusion with bovine serum albumin, egg albumin and pepsin was investigated by gel filtration chromatography and absorption spectra. The results demonstrated that prepared heme-β-cyclodextrin inclusion can bind proteins well.That is to say, heme-β-cyclodextrin inclusion may be a better formulation for immune adjuvant.