Research On Extraction Of Hemin By Protease From Pig Blood

Blood is an animal protein with high nutritive value and extensive resource, which can be widely applied in the food industry. Heme is kind of important natural iron porphyrin compound. It is the prosthetic group (active spot) in hemoglobins, the myoglobin and blood cells. Which widely exist in animals' blood, muscle and some plant organizations. It is also a natural pigment in animal blood and has important physiological functions and great use value.In the methods of heme determination, the spectrophotometry is a classical way for heme quantitative analysis. Its operation is simple and fast, the cost is low, is suitable for the massive sample determination. The HPLC has good separation effect, may combine with other instrument like mass spectrography. Because researchs on this aspect are quite few, it exists certain defects.Hemoglobin powder with pretreatment as raw material, the reaction selects some proteinase and compound enzymes to hydrolyze. The results indicate Alkaline proteinase has the best hydrolysis effect. The single factor and the orthogonal experiments give the best hydrolysis condition: temperature 60 oC, pH value 7.5, the substrate concentration 5% and enzyme/substrate 4%. In addition, different treatments of raw material and the compound-enzyme hydrolysis don't play obvious roles on the response.Centrifugalization is used to separate heme from the hemoglobin digest. The optimization craft is: after digest (30 oC) is centrifuged, wash the obtained sediment with water and centrifuge for 3 times, then all supernatants merge, adjust the pH value 4.0, centrifuge and wash sediment again for 3 times, freeze-drying the product. The product purity is more than 24.47% and yield is57.50%. Comparing the product with that of ultrafiltration (heme content 9.02%), effect of centrifugal separation is better than ultrafiltration.The research in product nature indicates, Product No.2 is amorphous powder, Product No.1 is pitchy massive; There is 0.1781g/g heme in Product No.1, while 0.2447 g/g in Product No.2. The results of ultraviolet scanning and gel chromatography shows, peptides and heme are contained in the digest, and peptide occupies the main part. The supernatants contain only peptides, and heme has already been removed. The Product No.2 has higher content of heme. The mass spectrography show: molecular weights of principal matter in product No.2 are 616.7 and 657, thus it should be monomer of heme and hemin. The two hold 70% part of the total heme .