Structural and functional characterization of proteolytic fragments derived from the C-terminal regions of bovine fibrinogen.
A number of new as well as previously described fragments derived from the D region of bovine fibrinogen by limited proteolysis have been characterized by sequence analysis, differential scanning calorimetry and circular dichroism. Determination of the extremities of the polypeptide chains forming individual fragments allowed the scheme of proteolysis and the borders between domains in the D region of fibrinogen to be established. It was also found that the most thermostable region of the D fragment (TSD) can be substantially reduced in size without loss of its compact structure. The alpha-helical content of the newly prepared 21-kDa TSD2 and 16-kDa TSD3 fragments were 82% and 75%, respectively, strongly supporting a coiled-coil structure for this region of the fibrinogen molecule. The DX and DZ fragments, prepared from a chymotryptic digest of the DLA fragment, were found to be similar to the DL and DY fragments, respectively, except for an internal cleavage at K393-T394 in their beta chains. This cleavage leads to destabilization of all thermolabile domains, indicating interaction between them. The DL and DY fragments, containing only one polymerization site in their beta chains, were able to inhibit fibrin polymerization at high concentration. However, these same fragments failed to bind to fibrin-Sepharose under conditions where their structural analogues, DX and DZ, were tightly bound, indicating that cleavage after K393 substantially increases the affinity of this site.
Litvinovich
S V
SV
J. Holland Laboratory, American Red Cross, MD 20855, USA.
Henschen
A H
AH
Krieglstein
K G
KG
Ingham
K C
KC
Medved
L V
LV
D000595:Amino Acid Sequence; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology*; D010447:Peptide Hydrolases / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D010446:Peptide Fragments; D005337:Fibrin; D005340:Fibrinogen; D010447:Peptide Hydrolases; D003488:Cyanogen Bromide