EVIDENCE FOR A CYSTEINE-HISTIDINE THIOETHER BRIDGE IN FUNCTIONAL UNITS OF MOLLUSCAN HEMOCYANINS AND LOCATION OF THE DISULFIDE BRIDGES IN FUNCTIONAL UNIT-D AND UNIT-G OF THE BETA(C)-HEMOCYANIN OF HELIX-POMATIA

In functional units d and g from the beta(C)-haemocyanin of the gastro pod Helix pomatia, aminoacid analysis in the presence of dimethyl sulf oxide showed the occurrence of seven cysteine residues. Titration with 5,5'-dithiobis(2-nitrobenzoic acid), however, did not reveal any free thiol group. Pepsinolysis at pH 2.0 followed by amino acid analysis a nd partial sequencing of the cysteine-containing peptides showed that six cysteine residues are involved in the formation of three disulfide bridges at corresponding positions. The results indicated that the re maining cysteine residue is linked by a thioether bridge to a histidin e residue located two positions further in the sequence (H. pomatia d Cys60 His62; H. pomatia g Cys66 His68). This residue corresponds to on e of the three histidine residues considered to be involved in the coo rdination of the copper A atom of the dinuclear copper group of the fu nctional units. The presence of the thioether bond was further evidenc ed by an absorption band at 255 nm and by molecular mass determination s with electrospray mass spectrometry on a peptic peptide from H. poma tia d and on peptides obtained by proteolysis of carboxymethylated H. pomatia d with trypsin and pronase. Upon sequence analysis the cystein e-histidine thioether bridge was cleaved into didehydroalanine (polyme rs) and 2-thiolhistidine. A peptide with a cysteine-histidine thioethe r bridge was isolated from pepsinolysates of functional units c, e, f, g and h of Sepia officinalis and unit g of Octopus vulgaris, obtained from haemocyanin of the cephalopods S. officinalis and O. vulgaris. A cysteine-histidine thioether bridge, which was reported previously fo r tyrosinase of Neurospora crassa, thus seems to be a general feature for functional units of molluscan haemocyanins.