Intersubunit fluorescence energy transfer in human factor VIII.
Human factor VIII circulates as a series of active heterodimers composed of a light chain (83 kDa) linked by divalent metal ion(s) to a variable sized heavy chain (93-210 kDa). Purified factor VIII subunits were modified with sulfhydryl-specific fluorophores. Probe selection was based upon the limited number of free cysteine residues in each subunit. Levels of probe incorporation suggested the presence of a single reactive cysteine residue per subunit. Amino-terminal sequence analysis of fluorescent tryptic peptides derived from the modified subunits indicated fluorophore attachment sites at Cys528 of the heavy chain (A2 domain) and Cys1858 of the light chain (A3 domain). Subunit reassociation was measured by fluorescence energy transfer using light chain modified with N-[1-pyrenyl] maleimide (fluorescence donor) and heavy chain modified with 7-diethylamino-3-[4'-maleimidophenyl]-4-methylcoumarin (fluorescence acceptor). Donor fluorescence quenching paralleled the formation of factor VIII clotting activity, and both effects were saturable with respect to added heavy chain. Based upon the degree of donor quenching, a distance of 20 A was calculated separating the two fluorophores. These results indicate a close spatial relationship between the A2 domain of heavy chain and the A3 domain of light chain in the factor VIII heterodimer.
Fay
P J
PJ
Department of Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
Smudzin
T M
TM
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides; D004735:Energy Transfer; D005169:Factor VIII / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds
D004220:Disulfides; D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; D005169:Factor VIII; D001120:Arginine; D008239:Lysine; D003545:Cysteine