Abstract
The state of aggregation of recombinant desulfatohirudin (r-HV1) in solution under physiological conditions (pH 7.5, 0.15N NaCl) was investigated by sedimentation equilibrium. The weight-average molecular weight ¯M w determined by sedimentation equilibrium was found to be 6914±76 Da compared to 6964 Da expected from the amino acid sequence. The ¯M z/¯M w ratio was found to be 1.03, which demonstrates that under the conditions studied hirudin exists in solution as a monomer. This result is in agreement with the relative molecular weight (M r) of recombinant hirudin variant 3 reported by Otto and Seckler [(1991),Eur. J. Biochem. 202, 67–73], who also used equilibrium ultracentrifugation, but not with the molecular weight estimated from gel permeation chromatography of natural hirudin (51,300 Da) [Konnoet al. (1988),Arch. Biochem. Biophys. 267, 158–166]. Knowledge of the state of aggregation is essential for understanding the mechanism of interaction of thrombin and hirudin under physiological conditions.
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Abbreviations
- ¯M w :
-
weight-average molecular weight
- ¯M z :
-
Z-average molecular weight
- M r :
-
relative molecular weight
- NTSB:
-
2-nitro-5-thiosulfobenzoic acid
- Tris:
-
Tris(hydroxymethyl)aminomethane
- r-HV1:
-
recombinant desulfatohirudin
- ∃ M :
-
molar extinction coefficient
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Thannhauser, T.W., Scheraga, H.A. State of aggregation of recombinant hirudin in solution under physiological conditions. J Protein Chem 15, 751–753 (1996). https://doi.org/10.1007/BF01887149
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DOI: https://doi.org/10.1007/BF01887149