Abstract

Blue oxygen binding protein hemocyanin from scorpion Buthus sindicus has been investigated using low resolution techniques. The native protein is a polymer of eight different types of subunits arranged in four cubic hexameric form (4x6-mers) as previously annotated using a combination of various types of chromatographic and electrophoretic techniques. In addition, both “top face” as well as the “side view” of the native assembly has also been identified from the negatively stained specimens using transmission electron microscopy confirming the overall structural features of arthropodan hemocyanins. These results are also supported from data obtained from another low resolution technique i.e. Small Angle X-ray scattering (SAXS). SAXS results under oxygenated and deoxygenated states represent a validation case for this technique with key conformational changes of Rg 88.0 → 86.0 Å ± 1% (Dmax 280.0 → 290.0 Å ± 2%), respectively suggesting that the oxygenated hemocyanin is longer then the deoxygenated hemocyanin by almost 2 Å. Likewise, active conformations of the purified structural and functional subunit Bsin1 under oxygenated and deoxygenated states also determined by SAXS measurements revealed a Rg value of 25.2 → 25.7 Å ± 1% (Dmax 75.0 → 75.5 Å ± 2%), respectively suggesting very little or no contribution of the individual subunit in the overall conformational change in the native assembly during molecular breathing. Preliminary molecular shapes for the oxy-molecules, calculated directly from the scattering profile-alone in a model-independent procedure, superimpose well on other closely related known threedimensional structures of the same size. Structural and functional aspects of the native as well as purified subunit and the application of these low resolution techniques like transmission electron microscopy and Small Angle X-ray scattering have been discussed.

Keywords: hemocyanin, low resolution techniques, oxygen transport protein, small angle x-ray scattering, scorpion, transmission electron microscopy