Structural characterization of very high density lipoprotein from the spider Polybetes pythagoricus

LAINO A,; GARCÍA F,; CUNNINGHAM M,; HERAS, H.

Va. Carlos Paz, Argentina

Congreso; XLIV Reunión SAIByBM; 2008

SAIByBM

We have previously isolated, purified and characterized an hemolymphatic VHDL from Polybetes pythagoricus. This lipoprotein contains hemocyanin (He) as the major apoprotein, and as a novelty, it transports most of the circulating lipids. In the present work , for the first time in arachnids , we studied size, shape and structure of P pythagoricus VHDL using electronic microscopy, MALDI -TOF-MS, circular dichroism, partial proteolysis , N-terminal sequencing, and lipid and copper gel specific staining. Results showed th at VHDL has spheroidal morphology with an estimated size of 11.2 =0.025 nm. As seen by SDS -PAGGE. It contains one main band corresponding to monomers of He (70 kDa) , and two min or subunits correspond ing to no n-respiratory prot eins of 105 and 120 kDa . These last two protein s are not linked by disulfide bonds. they don ´t include lipids or copper in the ir structure, and they would be more exposed to the aqueous medium in native conditions. Us ing circular dichroism we observed that it contains 20% a -helix, 29% B-sheet, 22.7% turns and 29.7% random. By MALDI-TOF MS, 15 polypeptides present in the monomer were found to be homologou s to subunit 3 of the spid er Cupiennius salei hemocyanin; and 27 polypeptides from the 105 kDa sub unit showed homology to a protein from the insect Anopheles gambiae .