The carbohydrate content of gastropod haemocyanins.

Gastropod haemocyanins are multisubunit copper-containing proteins with molecular weights in the region of 9 x lo6. They have been shown to be glycoproteins with total carbohydrate contents of 1-8 %, estimated by colorimetric and quantitative t.1.c. methods (Dijk et al., 1970). They are unusual in having no sialic acid residues, but, as is the case with most other glycoproteins, there are few clues as to the function, if any, of the sugar residues. To facilitate detailed analysis of glycopeptides derived from gastropod haemocyanins, for example, in order to investigate the carbohydrate composition as a possible source of the observed microheterogeneity in these proteins (Siezen &van Driel, 1973), it was necessary to use an efficient quantitative method for measuring individual monosaccharides in glycopeptides and glycoproteins. G.1.c. of trimethylsilyl derivatives of methylglycosides has been selected as the most appropriate method for this purpose. The haemocyanins studied in the present work were those from the whelks Buccinum undatum, Neptunea antiqua, Colus gracilis, the fresh-water snail Lymnaea stagnalis and the terrestrial snail Helixpomatia. These were extracted and purified as described previously (Heirwegh etal., 1961 ;Wood, 1973; Hall etal., 1975). When necessary the haemocyanins were stored under CO at 4°C in solution in 0.1 M-sodium acetate buffer, pH5.7, containing 0.01 % (w/v) NaN3. Methylglycosides derived from gastropod haemocyanins were prepared from 1-4mg of protein by the method of Bhatti et al. (1970). Mannitol was added as internal standard before methanolysis. Trimethylsilyl derivatives were prepared by adding 50p1 of trimethylsilyl reagent (pyridine/trimethylchlorosilane/hexamethyldisilazane, 5 : 1 : 1, by vol.) and dessicated over P,05 before chromatography. G.1.c. was carried out on a Pye 104 gas chromatograph fitted with a dual-flame ionization detector. A 75m open tubular column of internal diameter 0.25mm was used with OV 101 as liquid phase and He as carrier gas at a flow rate of 5 ml/min. Sample volumes of 0.1-1 .O,d were injected into the gas chromatograph. The temperature programme was as follows: 5min at 160°C, increase from 160°C to 200°C at 4"C/min, and finally lOmin at 200°C. Carbohydrate contents were estimated from the areas under the recorder trace by comparison with known monosaccharides. The response of this method is poor for amino sugars, which in any case produce asymmetric peaks. Amino sugar determinations were therefore carried out by ion-exchange chromatography on samples that had been hydrolysed with 4w-HCI in evacuated sealed tubes for 4h at 105°C. Samples for amino acid analysis were hydrolysed with ~ M H C I in evacuated sealed tubes at 105°C for 24, 48 and 96h. Amino sugar and amino acid analysis was performed on a Biocal amino acid analyser. Tryptophan was determined by the method of Spies & Chambers (1949), and cysteine was measured as cysteic acid in performic acid-oxidized samples (Leggett Bailey, 1962). The amino acid compositions of haemocyanins are known to be broadly similar, even when they originate from different phyla (Ghiretti-Magaldi et al., 1966; Dijk el al., 1970). Except for L. stagnalis, the haemocyanins investigated in the present work did not show great differences either amongst themselves or from other haemocyanins that have been analysed (Table 1). The haemocyanin of L . stagnalis had elevated concentrations of alanine and lysine, and a low histidine content compared with the other haemocyanins. As was observed by Witters & Lontie (1968), the concentration of methionine of H. pomatia haemocyanin was low, and the present work indicated that the same was true of L. stagnalis haemocyanin. Much more striking differences were observed in the sugar residue compositions. Whereas the marine prosobranch haemocyanins (B. undatum, N. antiqua and C. gracilis) possessed the sugars fucose, mannose, galactose, glucose, N-acetylglucosamine and N-acetylgalactosamine, the pulmonate-snail haemocyanins possessed, in addition, xylose and certain as yet unidentified sugars. The presence of an unidentified sugar in