Alpha- and beta-hemocyanin of Helix pomatia

This thesis deals with several aspects of the protein structure of Alpha- and Beta -hemocyanin of Helix pomatia. a- and 0-hemocyanin possess a virtually identical amino acid composition; it closely resembles the amino acid com- positions of hemocyanins of other MOLLUSCA and ARTHROPODA. All hemocyanins tested are glycoproteins; the carbohydrate content of Helix pomatia hemocyanin is among the largest of the investigated species of both phyla. Alpha- as well as Beta-hemocyanin has only one N-terminal amino acid: arginine. No C-terminal amino acid has definitely been identified; some evidence points to proline. Dissociation of hemocyanin into individual polypeptide chains is extremely difficult, due to the strong tendency to aggregate under all sorts of conditions. By applying a very sensitive method- polyacrylamide gel electrophoresis in SDSsubunits with a molecular weight of 25, 000 have been observed in formic-acid-treated hemocyanin. In ultracentrifugation experiments only dimers of these subunits were detected The molecular weight of 25,000 for the smallest subunit has been confirmed by quantitative determination of the N-terminal arginine. Several methods have been applied to obtain and separate the peptides of Helix pomatia hemocyanin. Digestion with trypsin and chymotrypsin yielded only slightly soluble peptide mixtures, which on column chromatography showed pronounced aggregation. From the products of cyanogen bromide cleavage only one peptide could be purified to some extent. Since the peptide lacks homoserine it must represent the C-terminal part of hemocyanin. The most successful approach appears to be the tryptic digestion of maleinylated hemocyanin. The completely soluble peptides may be separated on ion-exchange columns above pH 6; some preliminary results indicate that isolation of Nand C-terminal peptides from this digest will be relatively simple. Attempts were made to isolate the part of the molecule to which the carbohydrate moiety is attached. From a trypsinchymotrypsin and a subtilisin digest of a-hemocyanin glycopeptides were isolated. Although these glycopeptides could not be completely purified, due to their bad chromatographic properties, some conclusions may be drawn from the results of the analysis. The presence of at least two types of carbohydrate moiety was shown; it cannot be decided whether both types occur in the same subunit or in different ones. The carbohydrate moiety is attached to asparagine; a tentative amino acid sequence around the attachment site is proposed: -Ala -Asn-Ser -Thr-Gly - This sequence is in agreement with the sequence of the "recognition" site in other glycoproteins. It points to the presence of a complicated, branched carbohydrate chain; this is in agreement with the presence of not only glucosamine and mannose, but also fucose, galactose, glucose and xylose.