Structure of hemocyanin from garden snail Helix lucorum.
Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthropods with different quaternary structure. They are represented in the hemolymph of molluscs with one, two or three isoforms, as decameric, didecameric, multidecameric and tubules aggregates. We describe here the structure of the hemocyanin Helix lucorum (HlH), species in the series of molluscan hemocyanins. In contrast with other molluscan hemocyanins, three different hemocyanin isopolypeptides were isolated from the hemolymph of the garden snail H. lucorum, named as beta-HlH, alpha(D)-HlH and alpha(N)-HlH. Their molecular masses were determined by size exclusion chromatography to be 1068 kDa (beta-HlH) and 1079 kDa (alpha(D)-HlH, and alpha(N)-HlH). Native HlH exhibits a predominant didecameric structure as revealed by electron microscopy and additionally few tridecamers are shown in the electron micrographs of HlH resulting from the association of a further decamer with one didecamer. The three isoforms are represented mainly as homogeneous didecamers, but they have different behaviour after dissociation and reassociation in the pH-stabilizing buffer, containing 20 mM CaCl(2). All isoforms were reassociated into didecamers and tubules with different length, but in contrast to alpha(D)-HlH isoform, longer tubules were observed in beta-HlH. Moreover the structure of beta-HlH was analysed after limited proteolysis with trypsin followed by FPLC and HPLC separation of the cleavage products. Eight different functional units were identified by their N-terminal sequences and molecular masses. The protein characteristics, including UV absorption at 340 nm, fluorescence and CD spectra of the native molecule and its units confirmed the structure of multimer protein complexes.
Velkova
Ludmila
L
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev str bl.9, Sofia 1113, Bulgaria.
Dimitrov
Ivan
I
Schwarz
Heinz
H
Stevanovic
Stefan
S
Voelter
Wolfgang
W
Salvato
Benedeto
B
Dolashka-Angelova
Pavlina
P
D000595:Amino Acid Sequence; D000818:Animals; D006372:Helix, Snails / Q000737:chemistry*; D006433:Hemocyanins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D010446:Peptide Fragments; D021122:Protein Subunits; D006433:Hemocyanins