Studies on the Structure and Function of Myeloperoxidase from Normal Human Leukocytes

1. 髓过氧化物酶是从正常的人白细胞中纯化出来的，并首先结晶。结晶酶含有三种组分，通过阳离子交换层析分离得到三种组分。研究了这三种组分的分子量、分子形状、亚基结构、光吸收、圆二色性、磁性圆二色性和氨基酸组成。该酶由两个大亚基和两个小亚基组成，三种组分的大亚基分子量不同。还原后烷基化制备髓过氧化物酶半酶。半酶活性无明显变化。沉积-扩散和小角度x射线散射实验表明，全酶和半酶的分子形状比以前报道的更接近球形在盐酸胍存在下，酶还原后用色谱法分离出两种亚基及其N和c端周围的氨基酸序列。通过与髓过氧化物酶前体cDNA碱基序列的比较，确定了髓过氧化物酶前体被细胞蛋白酶加工的部位。此外，绿血红素通过共价键与大亚基蛋白结合。研究了两种加入过氧化氢后形成的髓过氧化物酶中间化合物的寿命和化学计量学。在反应过程中，证实了酶的真实过氧化氢酶活性。半酶具有与全酶相同的反应特性。

1. Myeloperoxidase was purified form normal human leukocytes, and it was first crystallized. The crystalline enzyme contained three components, which were isolated homogeneously by cation-exchange chromatograbhy.2. These three components were investigated on their molecular weight, molecular shape, subunit structure, light absorption, circular dichroism, magnetic circular dichroism, and amino acid composition. The enzyme consisted of two large subunits and two small subunits, and the three components were different in their molecular weight of the large subunits.3. The hemienzyme of myeloperoxidase was prepared by alkylation after reduction. The activity of the hemienzyme was not changed. The sedimentation-diffusion and small-angle X-ray scattering experiments showed that the molecular shape of the holo- and hemi-enzymes were more spherical than the values reported before.4 Two kinds of subunits were isolated by chromatography after the reduction of the enzyme in the presence of guanidine hydrochloride, and the amino acid sequences around their N- and C-termini. By comparing these sequences with those deduced from the cDNA base sequences for the precursor of myeloperoxidase, the processing part of the precursor by cellular proteinase was determined. Moreover, the green heme was found to be bound on the large subunit protein by covalent bonding.5. Two intermediate compounds of myeloperoxidase formed by the addition of hydrogen peroxide, and their life times and stoichiometry were investigated. In the course of the reaction, true catalase activity of the enzyme was confirmed. The hemienzyme has the same reaction characteristics of the holo-enzyme.