Affinity labeling of lysine-149 in the anion-binding exosite of human. alpha. -thrombin with an N sup. alpha. -(dinitrofluorobenzyl)hirudin C-terminal peptide

Journal Article · Tue Jul 10 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00479a006· OSTI ID:6253869

Bourdon, P; Maraganore, J M ^[1]; Fenton, II, J W ^[2]

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1. Biogen, Inc., Cambridge, MA (USA)
2. New York State Department of Health, Albany (USA)

In order to define structural regions in thrombin that interact with hirudin, the N{sup {alpha}}-dinitrofluorobenzyl analogue of an undecapeptide was synthesized corresponding to residues 54-64 of hirudin (GDFEEIPEEY(O{sup 35}SO{sub 3})L (DNFB-({sup 35}S)Hir{sub 54-64})). DNFB-({sup 35}S)Hir{sub 54-64} was reacted at a 10-fold molar excess with human {alpha}-thrombin in phosphate-buffered saline at pH 7.4 and 23{degree}C for 18 h. Autoradiographs of the product in reducing SDS-polyacrylamide gels revealed a single {sup 35}S-labeled band of M{sub r} {approximately}32,500. The labeled product was coincident with a band on Coomassie Blue stained gels migrating slightly above an unlabeled thrombin band at M{sub r} {approximately}31,000. Incorporation of the {sup 35}S affinity reagent peptide was found markedly reduced when reaction with thrombin was performed in the presence of 5- and 20-fold molar excesses of unlabeled hirudin peptide, showing that a specific site was involved in complex formation. The human {alpha}-thrombin-DNFB-Hir{sub 54-64} complex was reduced, S-carboxymethylated, and treated with pepsin. Peptic fragments were separated by reverse-phase HPLC revealing two major peaks containing absorbance at 310 nm. Automated Edman degradation of the peptide fragments allowed identification of Lys-149 of human thrombin as the major site of DNFB-Hir{sub 54-64} derivatization. These data suggest that the anionic C-terminal tail of hirudin interacts with an anion-binding exosite in human thrombin removed 18-20 {angstrom} from the catalytic apparatus.

• OSTI ID: 6253869
• Journal Information: Biochemistry; (USA), Vol. 29:27; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ENZYME INHIBITORS
AMINO ACID SEQUENCE
LYSINE
LABELLING
THROMBIN
MOLECULAR STRUCTURE
ALKYLATION
ENZYME ACTIVITY
LIQUID COLUMN CHROMATOGRAPHY
SERINE PROTEINASES
STOICHIOMETRY
SULFUR 35
AMINO ACIDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHROMATOGRAPHY
COAGULANTS
DAYS LIVING RADIOISOTOPES
DRUGS
ENZYMES
EVEN-ODD NUCLEI
HEMATOLOGIC AGENTS
HEMOSTATICS
HYDROLASES
ISOTOPES
LIGHT NUCLEI
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
RADIOISOTOPES
SEPARATION PROCESSES
SULFUR ISOTOPES
550201* - Biochemistry- Tracer Techniques