Abstract
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.
Highlights
Human factor VI11 circulates as a series of active ponent is minimally represented by the Al-A2 domains (93 heterodimers composed of a light chain (83kDa) linked kDa) but exhibits marked size heterogeneity resulting from by divalentmetal ion(s) toa variable sized heavy chain the presence of some or all of the contiguous B domain
Calculated separating the two fluorophores. These results indicate a close spatial relationship between the A2 domain of heavy chain and the A 3 domain of light chain in thefactor VI11 heterodimer
The isolated light and heavy chains of factor VI11 were reacted with the sulfhydryl-specific probes NPM and CPM, respectively
Summary
Human factor VI11 circulates as a series of active ponent is minimally represented by the Al-A2 domains (93 heterodimers composed of a light chain (83kDa) linked kDa) but exhibits marked size heterogeneity resulting from by divalentmetal ion(s) toa variable sized heavy chain the presence of some or all of the contiguous B domain. Transfection using a plasmid containinga contiguous 7.2kilobase factor VI11cDNA results insecretion of the separated reassociation was measured by fluorescence energy subunits that subsequently reassociate to generate active matransfer using light chain modified with N-[l-pyrenyl] terial [14]. These results indicate a close spatial relationship between the A2 domain of heavy chain and the A 3 domain of light chain in thefactor VI11 heterodimer. Purified subunits modified with sulfhydryl-specific fluorophores serve as the fluorescence donor/acceptor pairing inth: reconstituted protein.
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