This Article Full Text Full Text (PDF) Supplement All Versions of this Article: biophysj.106.100164v1 93/11/3963    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Banerjee, Y. Articles by Kini, R. M. Search for Related Content PubMed PubMed Citation Articles by Banerjee, Y. Articles by Kini, R. M.

Biophysical Characterization of Anticoagulant Hemextin AB Complex from the Venom of Snake Hemachatus haemachatus

Yajnavalka Banerjee ^* , Rajamani Lakshminarayanan  , Subramanian Vivekanandan ^* ¶, Ganesh Srinivasan Anand ^*, Suresh Valiyaveettil  and R. Manjunatha Kini ^* ||

^* Department of Biological Sciences, Faculty of Science, and Department of Chemistry, National University of Singapore, Singapore; Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California; School of Dentistry, Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, California; ^¶ Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, Singapore; and ^|| Department of Biochemistry, VCU Medical Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia

Correspondence: Address reprint requests to R. Manjunatha Kini, Protein Science Laboratory, Dept. of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117 543. E-Mail: dbskinim{at}nus.edu.sg.

Hemextin AB complex from the venom of Hemachatus haemachatus is the first known natural anticoagulant that specifically inhibits the enzymatic activity of blood coagulation factor VIIa in the absence of factor Xa. It is also the only known heterotetrameric complex of two three-finger toxins. Individually only hemextin A has mild anticoagulant activity, whereas hemextin B is inactive. However, hemextin B synergistically enhances the anticoagulant activity of hemextin A and their complex exhibits potent anticoagulant activity. In this study we characterized the nature of molecular interactions leading to the complex formation. Circular dichroism studies indicate the stabilization of β-sheet in the complex. Hemextin AB complex has an increased apparent molecular diameter in both gas and liquid phase techniques. The complex formation is enthalpically favorable and entropically unfavorable with a negative change in the heat capacity. Thus, the anticoagulant complex shows less structural flexibility than individual subunits. Both electrostatic and hydrophobic interactions are important for the complexation; the former driving the process and the latter helping in the stabilization of the tetramer. The tetramer dissociates into dimers and monomers with the increase in the ionic strength of the solution and also with increase in the glycerol concentration in the buffer. The two dimers formed under each of these conditions display distinct differences in their apparent molecular diameters and anticoagulant properties. Based on these results, we have proposed a model for this unique anticoagulant complex.