Domain structure of the Rapana thomasiana (Gastropod) hemocyanin

Sufficient and well-dosed oxygen supply is one of the prerequisites of human and animal life and nature has developed families of respiratory proteins to master this task: Hemoglobins' myoglobins (vertebrates), hemocyanins (molluscs, arthropods) and hemerythrins (worms). Though structurally and in molecular size completely different, these proteins have all in common that under physiological conditions, oxygen is reversibly bound to a central heavy metal ion From these three families, the hemocyanins are one of the largest and most complex biopolymers, known so far, reaching diameters of up to 25 nm, comparable to ribosomes and detailed primary, secondary and quaternary structure determination obviously belongs to one of the most tedious tasks in biochemistry. The subunits of molluscan hemocyanins are organized into series of globular folded regions. Their structure has been clearly resolved in the electron microscope as a string of seven or eight beads (the number depending on the source of the hemocyanin), each presenting a functional oxygen-binding unit Between each pair of functional units is a short flexible linker region consisting of 10-15 amino acid residues. Rapana thomasiana hemocyanin aggregates are constituted of two different types of subunits called RHSS1 and RHSS2 which were purified by ion exchange chromatography of the dissociated Rapana hemocyanin Various tri-, di- and mono-domain fragments of the subunit are obtained by "autolysis" and limited proteolysis with V8 (Glu-C) proteinase, trypsin and elastase. The individual fragments are isolated by ion exchange, gel and/or reversed-phase chromatography. Their molecular weights are determined by SDS electrophoresis and the N-terminal amino acid sequences analyzed by Edman degradation, thus revealing the precise ordering of the domains in the Ropana subunit. The results confirm the 8-functional unit structure. Comparison of the RHSS1 domain sequences with those of other gastropod hemocyanin subunits is made.

STANKA STOEVA ,WOLFGANG VOELTER ,PAVLINA DOLASHKA ,NICOLAY GENOV ,