State of aggregation of recombinant hirudin in solution under physiological conditions

The state of aggregation of recombinant desulfatohirudin (r-HV1) in solution under physiological conditions (pH 7.5, 0.15 N NaCl) was investigated by sedimentation equilibrium. The weight-average molecular weight MW determined by sedimentation equilibrium was found to be 6914 +/- 76 Da compared to 6964 Da expected from the amino acid sequence. The MZ/MW 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,) 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) [Konno et 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.