Background: Hemocyanins are oxyphoric blue copper-containing respiratory pigment present in the blood (hemolymph) of many species of arthropod and mollusc. Scorpion hemocyanin is composed of 2.4 monomeric subunits assembled in four hexameric blocks (4x6-mers). It contains eight different types of polypeptide chains present in two or four copies within the native molecule [l].
Methods: The work presented here deals with the characterization of different hemolymph proteins particularly oxygen carrier protein i.e. hemocyanin from scorpion B. sindicus. Many conventional as well as advanced fractionation techniques were used. Characterization of different hemolymph components was achieved. using electrophoretic, chromatographic, electron microscopic and spectroscopic techniques. Chemical characterization and N-terminal sequence analysis of the purified subunits was established using amino acid analyzer and automatic gas phase sequencer. Partial characterization of structurally and functionally important subunit Bsin 1 (72 -kDa) was carried out using a combination of MALDI-MS and automatic Edman degradation analysis after chemical and enzymatic fragmentations. Molecular conformation and low-resolution structural details of both native as well as purified subunit Bsin 1 was established by synchrotron solution X-ray :scattering and shape restoration in terms of spherical harmonics from scattering data alone. Preliminary crystallization conditions for subunit 8si:11 were also established.
Results and Discussion: Studies on scorpion Buthus sindicus hemocyanin shows that the native protein molecule is a 4x6-mers assembly as established by transmission electron microscopy, solution X-ray scattering and molecular shape analysis. These results are in good agreement with scorpion 4x6-mers model structure. Scorpion (B. sindicus) native hemocyanin consists -of eight different types of polypeptide chains or subunits out of which two subunits exist as non covalently linked heterodimer as determined by electrophoretic and chromatographic analysis. These subunits (B sinl-3) have been found to be highly heterogenous not only in their N-terminal amino acid sequence but also in terms of electrophoretic mobility in different systems, chromatographic behaviour and amino acid composition [2-3].
Although scorpions, spiders and horseshoe crabs belongs to the same subphylum (chelicerata), the thermal stability of scorpion hemocyanin is considerably lower than that of the tarantula (E. clifornicum) and horseshoe crab (E polyphemus) as has been observed by circular dichroism studies. Furthermore, copper-dioxygen system at the binuclear active site has a stabilizing effect and oxygenated forms of hemocyanins, are significantly more thermostable than the apo-forms [4-5]. This was also complemented by the effect of GnHCl denaturation OD the two conformational states of hemocyanin. The fluorescence properties of the IOcorpiol1 B. sindicus hemocyanin and their structural subunit Bsin 1 suggest, that the indole groups are in rather nonpolar environment, deeply "buried" in the interior of the native hemocyanin as well as ill the subunit Bsin l . This is in good agreement with primary and three,-dimensional structure of Limulus subunit II where the dinuclear copper-oxygen binding site has been reported to be buried in the core of domain two and surrounded by the completely conserved aromatic amino acids to avoid any irreversible oxidation in the micro environment of the active site .
Scorpion Buthus sindicus hemocyanin subunits Bsln2 and 3 possibly correspond to the A. australis subunits 5A and 3B as they indicate maximum N-terminal sequence homologies (79% & 64%, respectively). Whereas subunit Bsin 1 possibly corresponds to the A. australis subunit 3A in terms of its N-terminal anomaly towards Edman degradation. Higher sequence resemblance with other closely related subunits from L. polypbemus subunit II and E. californicum chain a, and X-ray scattering results for subunit Bsin l agree better with the T-state (corresponding to the oxygenated subunit Lpol-II) rather than with the R-state conformation, as apparent from their identical (25.0 A) Rg values [6-8].
Partial primary structure of structurally and functionally important subunit Bsin l (428 residues, 68%) was established using a combination of MALDI-MS and Edman degradation analysis. Comparison of sequence data with other closely related subunits L. polyphemus subunit II and E. californicum chain a shows close structural resemblance with subunit Bsin l (60% and 56%, respectively) suggesting a common structural and functional role. Sequence comparison between scorpion B. sindicus subul1it Bsin 1 and A. australis subunit A6  also shows 73% sequence which is not very surprising in view common phylogenetic origin.
Homology of their Some of the structurally important residues proposed for Limulus subunit II , mainly around the dinuclear copper-binding active sit e, calciumbinding site and the functionally important residues involved in the pseudo-twofold symmetry and the presume1 oxygen. entrance pathway, also proved to be strictly conserved in scorpion (B. sindicus) hemocyanin subunit Bsin 1. On the other hand, sequence variation around functionally important chloride-binding site  is different not only in the scorpions B. sindicus hemocyanin subunit Bsin l and A. australis A6 but also in the closely related E. californicum subunit a. Deviation in the primary structure related to the chloride-binding site suggest that the effect of chloride ions may vary in different hemocyanins.
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