Subunit heterogeneity in arthropod hemocyanins: I. Chelicerata
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- Markl, J., Markl, A., Schartau, W. et al. J Comp Physiol B (1979) 130: 283. doi:10.1007/BF00689845
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The hemocyanins of 2 spiders (Eurypelma californicum, Cupiennius salei), 2 scorpions (Pandinus pallidus, Androctonus australis), a whipscorption (Mastigoproctus brasilianus) and 2 whipspiders (Tarantula palmata, Trichodamon froesi) were analyzed for subunit heterogeneity by high resolution polyacrylamide electrophoresis (PAGE). For comparison,Limulus polyphemus hemocyanin was subjected to the same analytical scheme.
All of the species, except forLimulus (predominantly 60 S-hemocyanin) andCupiennius (16 S- and 24 S-hemocanin) possess only hemocyanin sedimenting in the 33 S to 37 S range. A second, major blood protein was observed in each species, safeMastigoproctus. This second, non-respiratory protein sediments with ca. 16 S, but with about 24 S in the case of the scorption species.
Upon incubation with sodium dodecylsulfate (SDS) and β-mercaptoethanol and electrophoresis in polyacrylamide gradients, between 2 and 5 hemocyanin bands are obtained, with average molecular weights between 70,000 and 75,000. The non-respiratory proteins yield two chains in each case, with molecular weights between 95,000 and 130,000.
Each hemocyanin could be dissociated at pH 9.6 to yield “native” subunits. By gel filtration, these were separated into monomers (4.5S) and dimers (6 S). No dimers were observed after dissociation ofPandinus and ofLimulus hemocyanin. Only in the case ofCupiennius the dimer is formed by a disulfide bridge. — PAGE of the dissociation mixture shows complex patterns which display 6–7 bands.
By preparative isolation of “native” subunits and subsequent analysis in SDS-PAGE, the two patterns of hemocyanin bands could be related to each other, and the total number of different polypeptide chains established with some certainty. It ranges from 5 inCupiennius 16 S-hemocyanin to 8 inAndroctonus hemocyanin and possibly 12 inLimulus hemocyanin. A loose correlation between oligomer size and number of different chains is suggested.