Ticu Elena-Loredana, Vercaigne-Marko Dominique, Huma Anca, Artenie Vlad, Toma Ovidiu, Guillochon Didier
Laboratorul de Biochimie, Facultatea de Biologie, Universitatea Al. I. Cuza, Iaşi, Romania.
Biotechnol Appl Biochem. 2004 Apr;39(Pt 2):199-208. doi: 10.1042/BA20030131.
The hydrolysis kinetics of native and denatured haemoglobin, using pepsin immobilized on aluminium oxide, was studied in order to produce hydrolysates containing bioactive peptides. Pepsin was immobilized on acidic alumina and on 2-ethanolamine- O -phosphate (2-EAOP)-modified acidic alumina. Surface charge of the two supports was determined as a function of pH. Kinetic studies were performed at 23 degrees C in 0.1 M acetate buffer, pH 4.5. At this pH, the surface charge of the two supports was almost the same. The coating of alumina by 2-EAOP only introduced a two carbon spacer between alumina surface and the reaction medium. Adsorption on the two supports of haemoglobin, haem and peptides produced in the course of hydrolyses were compared. Fixation of 2-EAOP on a pepsin-alumina complex gave hydrolysis kinetics of urea-denatured haemoglobin close to that obtained with the same amount of pepsin in solution, but with comparatively less adsorption of peptides and complete adsorption of haem. Heterogeneous hydrolyses of haemoglobin with pepsin, immobilized on functionalized alumina, resulted in the presence of VV-haemorphin-4, VV-haemorphin-7 and neokyotorphin in the supernatants without haem, the presence of which makes further purification difficult.
为了制备含有生物活性肽的水解产物，研究了使用固定在氧化铝上的胃蛋白酶对天然和变性血红蛋白的水解动力学。胃蛋白酶被固定在酸性氧化铝和2-乙醇胺-O-磷酸盐（2-EAOP）改性的酸性氧化铝上。测定了两种载体的表面电荷随pH的变化。动力学研究在23℃、pH 4.5的0.1 M醋酸盐缓冲液中进行。在此pH下，两种载体的表面电荷几乎相同。2-EAOP对氧化铝的包覆仅在氧化铝表面和反应介质之间引入了一个含两个碳的间隔基。比较了血红蛋白、血红素和水解过程中产生的肽在两种载体上的吸附情况。将2-EAOP固定在胃蛋白酶-氧化铝复合物上，得到的尿素变性血红蛋白水解动力学与相同量的溶液中的胃蛋白酶相近，但肽的吸附相对较少，血红素完全吸附。固定在功能化氧化铝上的胃蛋白酶对血红蛋白的非均相水解导致上清液中存在无血红素的VV-血啡肽-4、VV-血啡肽-7和新酪酪肽，它们的存在使得进一步纯化变得困难。
