State of aggregation of recombinant hirudin in solution under physiological conditions
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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.
Key words
Hirudin recombinant hirudin sedimentation equilibrium aggregationAbbreviations
- ¯Mw
weight-average molecular weight
- ¯Mz
Z-average molecular weight
- Mr
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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References
- Braun, P. J., Dennis, S., Hofsteenge, J., and Stone, S. R. (1988).Biochemistry 27, 6517–6522.PubMedGoogle Scholar
- Chang, J. Y. (1993).J. Biol. Chem. 268, 4043–4049.PubMedGoogle Scholar
- Chang, J. Y. (1994).Biochem. J. 300, 643–650.PubMedGoogle Scholar
- Chatrenet, B., and Chang, J. Y. (1992).J. Biol. Chem. 267, 3038–3043.PubMedGoogle Scholar
- Chatrenet, B., and Chang, J. Y. (1993).J. Biol. Chem. 268, 20988–20996.PubMedGoogle Scholar
- Chervenka, C., Chervenka, H. (1969).A Manual of Methods for the Analytical Ultracentrifuge, Beckman Instruments, Palo Alto, California, pp. 23–72.Google Scholar
- Grossenbacher, H., (1988).Information Sheet on Ciba-Geigy's Recombinant Hirudin, Ciba-Geigy Ltd., Biotechnology Department, K-681.2.43, CH-4002 Basle, Switzerland.Google Scholar
- Harvey, R. P., Degryse, E., Stefani, L., Schamber, F., Cazenave, J.-P., Courtney, M., Tolstoshev, P., and Lecocq, J.-P. (1986).Proc. Natl. Acad. Sci. USA 83, 1084–1088.PubMedGoogle Scholar
- Konno, S., Fenton, J. W., Villanueva, G. B. (1988).Arch. Biochem. Biophys. 267, 158–166.PubMedGoogle Scholar
- Otto, A., and Seckler, R. (1991).Eur. J. Biochem. 202, 67–73.PubMedGoogle Scholar
- Stone, S. R., and Hofsteenge, J. (1986).Biochemistry 25, 4622–4628.PubMedGoogle Scholar
- Stone, S. R., and Maraganore, J. M. (1993).Meth. Enzymol. 223, 312–336.PubMedGoogle Scholar
- Tertrin, C., de la Llosa, P., and Jutisz, M. (1966).Biochim. Biophys. Acta. 124, 380–388.PubMedGoogle Scholar
- Thannhauser, T. W., and Scheraga, H. A. (1997).Biochemistry, submitted.Google Scholar
- Thannhauser, T. W., Konishi, Y., and Scheraga, H. A. (1984).Anal. Biochem. 138, 181–188.PubMedGoogle Scholar
- Tuong, A., Maftouh, M., Picard, C., and Gachon, M. (1990).Anal. Biochem. 189, 186–191.PubMedGoogle Scholar
- Willard, H. H., Merritt, Jr., L. L., Dean, J. A., and Settle, Jr., F. A. (1988).Instrumental Methods of Analysis, Wadsworth, Belmont, California, pp. 644–649.Google Scholar