【作者】 张小芬；

【导师】 李任强；

【作者基本信息】 暨南大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 由于血红素（heme）、含血红素蛋白质（heme-containing protein）在生物学、医学和环境科学等的研究和应用中倍受关注，是目前研究的热点之一。本课题在血红素研究的最新进展上，对血红素应用于生物化学分析进行了研究。 过氧化氢及血清中葡萄糖的检测在临床生物化学上具有重要意义。用惰性载体Sepharose 4B、微晶纤维素、人造沸石、琼脂糖等分别直接固定氯化血红素（hemin），装柱后应用于痕量过氧化氢的检测，同时利用葡萄糖氧化酶偶联模拟过氧化物酶法用于血清葡萄糖的检测，并比较它们的检测灵敏度。实验结果表明，以上几种载体经活化后均能很好地固定血红素，固定化的血红素装柱后保持稳定的催化过氧化氢的活性，检测灵敏度高。血红素在几种载体上检测灵敏度的大小为：Sepharose 4B＞琼脂糖＞微晶纤维素＞人造沸石。本法操作简单且成本低，固定化的血红素反应柱可反复使用，能快速检测样品中的痕量过氧化氢及大量血样中的葡萄糖。 鉴于血清白蛋白分子的活泼性，在体外将牛血清白蛋白（bovine serum albumin，BSA）与氯化血红素以相同摩尔量结合制成血红白蛋白（hemalbumin），测定了其过氧化物酶活性。实验结果表明，血红白蛋白具有过氧化物酶特性，是一稳定而又活泼的分子。将血红白蛋白固定在已活化的Sepharose 4B载体上制成血红白蛋白柱子，偶联率达每克载体结合5.3mg血红白蛋白。将血红白蛋白柱子应用于测定痕量过氧化氢，显示正常、稳定的过氧化物酶活性，具极高测定灵敏度，能明显的测出低至3.63×10^-7mol/L浓度的过氧化氢。实验进一步探讨了人工合成的血红白蛋白用于催化H₂O₂氧化对甲基酚的反应体系，研究了含血红素白蛋白的过氧化物酶特征及其催化动力学特性，稳态速率法测定了米氏常数（K_m）、米氏速率（V_m）及反应级数等动力学参数。讨论了以甲基酚作为血红白蛋白氢供体底物的酶催化反应机理，确立了反应速率方程。实验发现血红白蛋白作为过氧化物酶（HRP）的替代物，其催化活性比氯化血红素、β-环糊精-氯化血红素（β-CD-hemin）等模拟过氧化物酶要高。 Sepharose 4B经环氧氯丙烷活化后直接固定氯化血红素制备亲和色谱柱，载体与氯化血红素的偶联率十分高，即每g溶胀的Sepharose 4B能固定高于0.25mg更多还原

【Abstract】 Studies on heme and heme-containing protein are hot topics due to their broad application in biology, medicine and environmental science, this paper has described the studies on biochemical analysis with hemin based on the recent advance of heme.The determinations of trace hydrogen peroxide and glucose in serum are valuable in clinic biochemistry. Sepharose 4B, agarose, cellulose microcrystalline and permutite was respectively used as the vehicle to immobilize hemin to make the columns, which were applied to measure trace hydrogen peroxide. Furthermore, glucose concentration in serum was measured by the method of glucose oxidase（GOD） conjugating mimic enzyme, and the measurement sensitivity of these columns were examined. Results showed that hemin may be immobilized well by these activated vehicles. The hemin columns presented a stable activity of hemin and a high measurement sensitivity, but the measurement sensitivity of hemin was different in different vehicles as following: Sepharose 4B> agarose > cellulose microcrystalline > permutite. Manufacture of hemin column was cheap, the column may be used repeatedly and applied for quick examination of trace hydrogen peroxide or glucose concentration in serum.Equimolar BSA and hemin were combined in vitro to form hemalbumin based on the vivid property of bovine serum albumin（BSA）, of which the peroxidase activity was examined . Results showed that hemalbumin possessed the peroxidase property, and it was a stable, active molecule. Then hemalbumin was immobilized to the activated vehicle Sepharose 4B with the couple ratio of 5.3mg hemalbumin per gram vehicle to make hemalbumin column, which was applied to measure trace hydrogen peroxide. Hemalbumin column presented a high measurement sensitivity, a stable peroxide as low as 3.63×10^-7mol/L. This experiment went step further to explore that synthesized hemalbumin was used in the catalytic oxidation of p-cresol by hydrogen peroxide. The peroxidase characteristics of hemalbumin and enzymatic kinetics were studied. The K_m value(1.85×10^-3mol/L) and V_m value(1.6min^-1) were measured by steady-state catalytic velocity at pH=10.3 and temperature 25℃, The reaction mechanism was discussed when p-cresol was used as a hydrogen donor for hemalbumin. The velocity equation更多还原