Determination of the disulfide bridges in factor Va heavy chain

Abstract

The M(r) = 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 A1 domain contains four cysteines while the A2 domain contains six cysteines. The locations of disulfide bridges and 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, free cysteines were identified by the incorporation of radioactivity while disulfide-containing 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, Cys139-Cys165 and Cys220-Cys301. One disulfide bond was explicitly identified in the A2 domain; Cys471-Cys497, and a free cysteine was found in the A2 domain at Cys538. Significant difficulties were encountered in preparing identifiable or soluble peptides which would permit the explicit identification of the three remaining cysteines in the A2 domain. On the basis of homology, it is likely that Cys589 is a free SH while a disulfide bridge exists between Cys579 and Cys660. Thus, three major disulfide bonding patterns, characterized as "alpha", "beta", and "gamma" loops, are found in factor V. Each A domain contains a 26 residue "alpha loop at positions 139-165, 471-497, and 1684-1710. The A1 and A2 domains each contain 81 amino acid residue "beta" loops at 220-301 and 579-660.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor V: a combination of Dr Jekyll and Mr Hyde

“…39 Factor V has 19 cysteine residues (Table 1 and Figure 1A show locations). 41,42 Five of the cysteines are present as free ϪSH, whereas the remaining 14 are involved in disulfide bridges forming several loops: three 26 amino acid residue ␣-loops are present, one in each A domain; the A1 and A2 domains each contain one ␤-loop that is composed of 82 amino acids (Table 1); 2 ␥-loops that are composed of 154 and 155 residues, that represent the C1 and C2 domains ( Figure 1A) each with a free ϪSH. The cysteine residue at position 539 appears to be very reactive.…”

“…Activated protein C resistance may also be observed with elevated levels of homocysteine. 149,150 Factor V contains 5 unpaired cysteines, 2 in the heavy chain, 2 in the light chain, and 1 in the B region 41,42 (Table 1). These cysteines can incorporate homocysteine at physiologically relevant concentrations associated with hyperhomocystinemia and, as a consequence, display a form of acquired APC resistance.…”

Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A)

“…The disulfide bridge and free cysteine shown as a dotted line were not determined, but proposed. Data are taken from Xue et al (1993Xue et al ( , 1994 and Takahashi et al (1984) for factor V and ceruloplasmin, respectively.…”

“…Most of the half-cystines present in the heavy and light chains are located in positions similar to those in factor V and ceruloplasmin. The locations of the disulfide bonds and free cysteines in the heavy (Xue et al, 1994) and light chain (Xue et al, 1993) of factor V and ceruloplasmin (Takahashi et al, 1984) have been established. Similar disulfide bond structures have been predicted in factor VI11 from the sequence homology between factor VI11 and factor V (Xue et al, 1993(Xue et al, , 1994.…”

“…The locations of the disulfide bonds and free cysteines in the heavy (Xue et al, 1994) and light chain (Xue et al, 1993) of factor V and ceruloplasmin (Takahashi et al, 1984) have been established. Similar disulfide bond structures have been predicted in factor VI11 from the sequence homology between factor VI11 and factor V (Xue et al, 1993(Xue et al, , 1994. It is important, however, to determine the location of the disulfide bonds in human factor VI11 to define further the structure/function relationship of this essential blood coagulation factor.…”