Characterization of domains obtained from a mollusc haemocyanin by limited proteolytic digestion.
The haemocyanin from the freshwater gastropod Lymnaea stagnalis was digested with proteolytic enzymes under conditions where it existed as whole (native) molecules (mol.wt. approx. 9 X 10(6)), or as one-tenth molecules. Digestion of whole molecules yielded a fragment of mol.wt. approx. 110,000 believed to correspond to the 'collar' of the molecule, and an aggregate some 20--30 times the size of the original native molecule formed by end-to-end polymerization of the molecule after removal of the collar. Digestion of one-tenth molecules yielded a mixture of products that could be separated into three fractions by gel filtration. Analysis of these by sodium dodecylsulphate/polyacrylamide-gel electrophoresis revealed that they typically contained two or three components. The collar fragment was present as a component of the intermediate-molecular-weight fraction, and it dissociated on sodium dodecyl sulphate/polyacrylamide gels to give two bands corresponding to apparent mol.wts. 65,000 and 60,000. The c.d. spectra of the separated fractions were recorded and fitted with Gaussian curves by a computer procedure. The fractions each possessed distinct c.d. spectra, by which they could be identified: the collar-fragment c.d. and absorption spectra showed the most striking differences compared with those of the other fragments. The results were interpreted in terms of the postulated existence, within the haemocyanin molecule, of multi-domain structures, each comprising a single polypeptide chain of mol.wt. 200,000--300,000.
D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006433:Hemocyanins; D008195:Lymnaea / Q000032:analysis*; D008854:Microscopy, Electron; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin
D010446:Peptide Fragments; D006433:Hemocyanins; D014357:Trypsin