Date on Master's Thesis/Doctoral Dissertation

3-2013

Document Type

Senior Honors Thesis

Department

Chemistry

Author's Keywords

Blood clotting/coagulation; Factor XIII; Thrombin; Fibrin; Alpha-C domain

Abstract

In the blood coagulation cascade, Factor XIII (FXIII) is a zymogen that is activated by thrombin (IIa) and Ca2+. Activated FXIII (FXIIIa) functions as a transglutaminase in the final steps of the clotting cascade by forming covalent crosslinks between fibrin monomers at reactive glutamine residues. This forms a stable clot structure, secure from proteolytic degradation. To monitor FXIIIa substrate sequence specificity, the glutamine-containing peptide models S. aureus Fnb A (100-114), α2AP(Q4P) (1-15) and K9 (1-10) were introduced to the enzyme in the presence of 15NH4Cl. Using MALDI-TOF MS and 15N HSQC NMR experiments, it was seen that α2AP(Q4P) (1-15) and K9 (1-10) were viable FXIIIa substrates, having isotopic nitrogen incorporated in their side chain amide groups. However, S. aureus Fnb A (100-114) was not able to undergo this catalytic reaction because of an interfering side reaction with the labeled nitrogen. The substrate specificity appears to be the result of chemical environment provided by the residues immediately surrounding the reactive glutamine, with a focus on the hybridization of the side chain carbons. The αC(233-425) domain of fibrin monomers was also analyzed as an intact FXIIIa substrate protein. The reactive Q328 residue of the protein was seen to undergo enzymatic transglutamination linearly with time using MALDI-TOF MS. 2D 15N HSQC experiments displayed that Q328 was not the only reactive glutamine in the protein but rather one of two or three reactive glutamines.

Included in

Chemistry Commons

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