Determination of the disulfide bridges in factor Va heavy chain

J Xue, M Kalafatis, JR Silveira, C Kung, KG Mann - Biochemistry, 1994 - ACS Publications
J Xue, M Kalafatis, JR Silveira, C Kung, KG Mann
Biochemistry, 1994ACS Publications
Revised Manuscript Received June 29, 1994® abstract: The M,= 94 000 heavy chain of
bovine factor Va contains 10 cysteine residues which are distributed in the 2 A domains
which make up this portion of the factor V molecule. The Al domain contains four cysteines
while the A2 domain containssix cysteines. The locations of disulfide bridgesand free
cysteines in bovine factor Va heavy chain were analyzed using iodo [14C] acetamide-
labeled factor Va heavy chain digested with trypsin, plasmin, V-8 protease, and cyanogen …
Revised Manuscript Received June 29, 1994® abstract: The M,= 94 000 heavy chain of bovine factor Va contains 10 cysteine residues which are distributed in the 2 A domains which make up this portion of the factor V molecule. The Al domain contains four cysteines while the A2 domain containssix cysteines. The locations of disulfide bridgesand free cysteines in bovine factor Va heavy chain were analyzed using iodo [14C] acetamide-labeled factor Va heavy chain digested with trypsin, plasmin, V-8 protease, and cyanogen bromide. Following HPLC separation of the resulting peptides, freecysteines were identified by the incorporation of radioactivity while disulfidecontaining peptides were detected using an SBD-F fluorometric assay after reduction. All cysteine-containing peptides were analyzed by amino acid sequence analysis. The four cysteines in the A1 domain are associated with two disulfide bonds, Cysi39-Cysi65 and Cys22o-Cys3oi. One disulfide bond was explicitly identified in the A2 domain; Cys47i-Cys497, and a free cysteine was found in the A2 domain at Cys53s. Significant difficulties were encountered in preparingidentifiable or soluble peptides which wouldpermit the explicit identification of the three remaining cysteines in the A2 domain. On the basis of homology, it is likely that
Cyssgg is a free SH while a disulfide bridge exists between Cys579 and Cys660'Thus, three major disulfide bonding patterns, characterized as “a”,“0”, and “7” loops, are found in factor V. Each A domain contains a 26 residue “a loop at positions 139-165,471-497, and 1684-1710. The Al and A2 domains each contain 81 amino acid residue “/3” loops at 220-301 and 579-660. The C domains contain 2 “7” loops of 154 (Cl) and 155 (C2) residues, respectively, 1866-2020 and 2025-2180.
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