Permissible discontinuity region of the alpha-chain of hemoglobin: noncovalent interaction of heme and the complementary fragments alpha 1-30 and alpha 31-141.
Permissible discontinuity region of the alpha-chain of hemoglobin: noncovalent interaction of heme and the complementary fragments alpha 1-30 and alpha 31-141.
复制标题
血红蛋白 α 链允许的不连续区域：血红素与互补片段 α 1-30 和 α 31-141 的非共价相互作用。
DOI：
10.1021/bi00368a017
复制
发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Acharya,AS
中科院分区：
文献类型：
--
作者：
Seetharam,R;Dean,A;Iyer,KS;Acharya,AS
Materials and MethodsHbA and the p-hydroxymercuribenzoate (HMB) reacted «-chain of HbA were prepared as described previously (Ac-harya & Manning, 1980). Staphylococcus aureus V8 protease was obtained from either Miles Laboratories or Worthington Biochemicals. Seppak cartridges were procured from Water Associates. Sephadex was purchased from Pharmacia. Centricon 10 microconcentrators were obtained from Amicon. Digestion of a-Chain by V8 Protease.--chain in the carbonmonoxy form (0.4 mM in 10 mM KH2P04, 1 mM EDTA, pH 6.0) was incubated with V8 protease (enzyme:«-chain ratio, 1: 250) at 37, 25, or 4 C. After the desired period of incubation, the digests were either subjectedto RPHPLC or Sephadex G-50 gel filtration. HPLC. The HPLC system used was assembled in the laboratory from commecially available components (Acharya et al., 1981). The samples were desalted by passing them through Seppak cartridges previouslyequilibrated with 0.1% TFA. Globin, heme, and the peptides were bound to Seppak under these conditions. The column was washed with 10 mL of 0.1% TFA. The 0.1% TFA eluate contained only the buffer salts. The material bound (globin, heme, and peptides) to the Seppak cartridge was eluted with about 1.5-2.0 mL of 70% acetonitrile containing 0.1% TFA and was isolated by lyophilization. The dried material was dissolved in 400 pL of 0.5% TFA and loaded on a Whatman Partisil 10 ODS-3 column. This procedure using Seppak gave consistently high yields (> 90%) of globin, globin fragments, and heme, free of buffer salts. The procedures for the preparation of globin, amino acid, and sequence analysis have been described earlier (Seetharam, 1983; Acharya & Seetharam, 1985). Sephadex G-50 Gel Filtration. A Sephadex G-50 column (2.0 X 60 cm) equilibrated with 10 mM KH2P04 and 1 mM EDTA, pH 6.0, was used in these studies. Ultrafiltration. These were carried out with either a regular ultrafiltration cell or Centricon 10 microconcentrators, de-pending on the volume to be concentrated. ResultsProteolysis of a-Chain with Staphylococcus aureus V8 Protease. V8 protease has two pH optima, one around pH 4.0 and the other around pH 8.0. Around pH 6.0, the specific activity of the enzyme is the lowest. We have chosen this pH for the digestion of the «-chain with the objective of limiting the cleavage to one of the glutamic acid residues of the «-chain. A kinetic analysis of the digestion of the «-chain by protease at 37 C (enzyme: substrate ratio, 1: 250) as revealed by RPHPLC analysis is shown in Figure 1. The chromatograms shown were obtained with samples of the «-chain that had been digested with V8 protease for*/2, 2, and 3 h, respectively, and are qualitatively similar. Each of them contains at least five chromatographically distinct components, designated, in the order of their elution,«Vb «V2,«V3,«V4, and «V5, respectively. Chromatography of the undigested «-chain showed that the «V5 region corresponds to the elution position of heme and the undigested-globin. It may be seen that with theprogress of digestion, a shoulder appears in the 3-h digest and a com-ponent separates partially from the-globin peak region.
