Kraehenbuhl J P, Galardy R E, Jamieson J D
J Exp Med. 1974 Jan 1;139(1):208-23. doi: 10.1084/jem.139.1.208.
A heme-octapeptide (mol wt 1,550) has been obtained from cytochrome c by successive pepsin and trypsin hydrolysis and purified by gel filtration and countercurrent distribution. It possesses peroxidatic activity characterized by an apparent K(m) of 0.2 M, an apparent v(max) of 4 mmol/min per mg of peptide, and a pH optimum of 7.0. Using a novel two-step conjugation procedure, the heme-octapeptide was coupled to rabbit Fab antibody fragments by first derivatizing it with the N-hydroxysuccinimide ester of p-formylbenzoic acid and subsequently allowing it to form a Schiff base with the amino groups of Fab. Stable covalent linkages were then obtained by reduction of the Schiff bases with sodium borohydride. The conjugate consists of approximately 2 heme-octapeptides attached to each Fab molecule. The molecular weight is 45,000 daltons when coupled to sheep Fab and 50,000 daltons with a Stokes radius of 32 A, when conjugated to rabbit Fab. Its peroxidatic activity is characterized by an apparent K(m) of 0.4 M, an apparent v(max) of 0.4 mmol/min and per mg of attached heme-octapeptide and a pH optimum of 7.0. The conjugate has been used for the localization at the electron microscope level of secretory immunoglobulins in the mammary gland of lactating rabbits.
通过胃蛋白酶和胰蛋白酶的连续水解从细胞色素c中获得了一种血红素八肽（分子量1550），并通过凝胶过滤和逆流分配进行纯化。它具有过氧化物酶活性，其表观米氏常数（K(m)）为0.2 M，表观最大反应速度（v(max)）为每毫克肽4 mmol/min，最适pH为7.0。采用一种新颖的两步偶联方法，首先用对甲酰苯甲酸的N-羟基琥珀酰亚胺酯对血红素八肽进行衍生化，随后使其与Fab的氨基形成席夫碱，从而将血红素八肽与兔Fab抗体片段偶联。然后用硼氢化钠还原席夫碱，得到稳定的共价键。该偶联物由每个Fab分子连接约2个血红素八肽组成。与羊Fab偶联时分子量为45000道尔顿，与兔Fab偶联时分子量为50000道尔顿，斯托克斯半径为32 Å。其过氧化物酶活性的表观米氏常数为0.4 M，表观最大反应速度为每毫克连接的血红素八肽0.4 mmol/min，最适pH为7.0。该偶联物已用于在电子显微镜水平定位泌乳兔乳腺中的分泌型免疫球蛋白。
