Abstract
Annelid hemoglobins are organized in a very complex supramolecular network of interacting polypeptides, the structure of which is still not wholly resolved. We have separated by two-dimensional electrophoresis the 4-MDa chlorocruorin of Sabella spallanzanii and identified its components by amino-terminal sequencing. This work reveals a high rate of heterogeneity of constituent chains in a single animal as well as in the Sabella population. Using a cDNA library prepared from the hematopoietic tissue of this worm, we have isolated and fully sequenced most globin and linker cDNAs. The primary structure features of these polypeptides have been characterized by comparison with model globin and linker sequences.
Highlights
Chlorocruorins (Chls)1 are giant extracellular oxygen-binding heme proteins found in four marine polychaete families
All hexagonal bilayer (HBL) Hbs and Chls are composed of two type of chains, heme-containing 16 –17-kDa globin chains and nonglobin linker chains of 25–32 kDa in an approximate 2:1 molar ratio [1]
A complex pattern of multiple spots is clearly detectable. They can be divided into three groups: heavy linkers, light linkers, and globin chains (Glb)
Summary
Purification and Gel Separation of S. spallanzanii Globin and Linker Chains—Live specimens of S. spallanzanii were collected in the Bay of Napoli. After checking the purity by a second SDS-polyacrylamide gel electrophoresis run, the purified proteins were emulsified with complete Freund’s adjuvant and injected into rabbits. Two rounds of subscreening were performed to select single positive clones Both strands of the cDNA inserts were sequenced by a primer-walking strategy using the fluorescent BigDyeTM terminators chemistry (PE Biosystems), and the sequencing reactions were analyzed on an ABI-377 automated DNA sequencer (PE Biosystems). The same protocol was adopted for the isolation of globin 3 cDNA, because immunoscreening identified no positive clones In this case, a degenerated reverse primer coupled to the BstXI adaptor primer was designed on the amino terminus of globin D. The pI and molecular mass were computed from the cDNA sequences with the program Compute pI/Mw and compared with the experimental data obtained from the 2-DE gel analysis. The linker chains were aligned using the ClustalW program [28], and the globins were manually aligned according to the tertiary structure template of invertebrate globins [19]
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