Determination of the disulfide bridges in factor Va heavy chain.
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)
Xue
J
J
Department of Biochemistry, University of Vermont, College of Medicine, Burlington 05405-0068.
Kalafatis
M
M
Silveira
J R
JR
Kung
C
C
Mann
K G
KG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D015943:Factor Va / Q000737:chemistry* / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D005456:Fluorescent Dyes; D005464:Fluorobenzenes / Q000737:chemistry; D005470:Fluorometry; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D007457:Iodine Radioisotopes; D013439:Sulfhydryl Reagents; D015943:Factor Va; C037802:7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D005341:Fibrinolysin; D003545:Cysteine; D003488:Cyanogen Bromide