Quaternary Structure of the Extracellular Haemoglobin of the Lugworm Arenicola marina

Abstract

To elucidate the quaternary structure of the extracellular haemoglobin (Hb) of the marine polychaete Arenicola marina (lugworm) it was subjected to multi-angle laser-light scattering (MALLS) and to electrospray-ionisation mass spectrometry (ESI-MS). It was also subjected to SDS/PAGE analysis for comparative purposes. MALLS analysis gave a molecular mass of 3648 % 24 kDa and a gyration radius of 11.3 rt 1.7 nm. Maximum entropy analysis of the multiply charged electrospray spectra of the native, dehaemed, reduced and carbamidomethylated Hb forms, provided its complete polypeptide chain and subunit composition. We found, in the reduced condition, eight globin chains of molecular masses 15952. Keywords: Arenicola ; haemoglobin ; quaternary structure ; multi-angle laser-light scattering; electrospray mass spectrometry.The giant extracellular haemoglobins (Hb) and chlorocruorins found in annelids [1] and vestimentiferans [2, 31 are characterised by an acidic isoelectric point and by an hexagonal symmetry in electron micrographs consisting of two superimposed hexagonal arrays of twelve spherical subunits, the hexagonal bilayer (HBL). These HBL Hb also contain a low haem and iron contents (about 67% of those observed in other Hbs) and consist of two types of chains, globin chains (~1 6 -1 8 kDa), accounting for approximately 70% of the total mass, and haem-deficient linker chains (~2 4 -2 8 kDa) necessary for the assemblage into the HBL structure [1,. The intertidal marine polychaete Arenicola marina (L.) was one of the first 60s (~3 6 0 0 kDa)

Recent New Characterizations on the Giant Extracellular Hemoglobin of Glossoscolex paulistus and Some Other Giant Hemoglobins from Different Worms

“…Besides, very recent preliminary work on the crystal structure of HbGp [21] has also suggested a strong similarity between HbGp and HbLt, both belonging to class 1, where the two hexagonal layers forming the bilayer are rotated to 16 degree one relative to another. It is worthy of mention, that another extracellular hemoglobin from a sea worm, HbAm, has also been investigated by Zal et al [22,23]. In this case the structure belongs to class 2, where the two hexagonal layers are exactly eclipsed one relative to the other, as demonstrated in recent crystallographic studies at low resolution [24].…”

Electrospray Ionization Mass Spectrometric Determination of the Molecular Mass of the ∼200-kDa Globin Dodecamer Subassemblies in Hexagonal Bilayer Hemoglobins

“…For the Arenicola Hb subassembly, there are 4 possible combinations of 3M from two different monomers (chains a1 and a2) and 35 possible combinations of 3T from five different trimers, leading to 140 possible M 3 T 3 subassemblies. Because the proportions of the subunit isoforms are known experimentally from previous ESI-MS studies (8,12), it is possible to calculate a mass distribution based on a straightforward combinatorial model (see "Appendix"). The calculated histograms for the subassembly masses of the two Hbs incorporating only masses with a probability Ն 0.002 are shown in Fig.…”

“…Arenicola Hb contains eight different globin chains (a1, a2, b1, b2, b3, c, d1, and d2) ranging in mass from 15,922 to 17,032 Da, with the b, c, and d chains forming five of the six possible disulfide-bonded trimers (T1-T5) (c ϩ b1 ϩ d1, c ϩ b1 ϩ d2, c ϩ b2 ϩ d1, c ϩ b2 ϩ d2, and c ϩ b3 ϩ d2) (12). Table I shows the range of masses observed for the two subassemblies and the selected representative masses together with the calculated masses for dodecamer subassembly compositions M 9 T, M 6 T 2 , and M 3 T 3 and a hexadecamer subassembly M 4 T 4 using the globin chain and subunit masses determined previously (8,12). For the Lumbricus Hb subassembly, the mass 214,190 Da at a de-clustering voltage of 140 V was taken to be representative, assuming a behavior similar to that of the Arenicola Hb subassembly, which displays minimal heme loss at this voltage.…”

“…as described elsewhere (12). The Hbs were dissociated at a neutral pH in the presence of ϳ4 M urea and subjected to low-pressure, isocratic gel filtration at room temperature (20 Ϯ 2°C) using a fast protein liquid chromatography system (Amersham Pharmacia Biotech) and 2.5 ϫ 50-cm columns of Superose S12 or S6 (Amersham Pharmacia Biotech).…”

“…The flow rate was 0.5 ml/min, and the absorbance of the eluate was monitored at 280 nm. The concentrations of the Hb and the globin subassembly were determined from the absorbance of the cyanmet forms at 540 nm using the extinction coefficients 0.442 and 0.656 ml⅐mg Ϫ1 ⅐cm Ϫ1 , respectively (12,26). Electrospray Ionization Mass Spectrometry-Sample solutions in 10 mM ammonium acetate were introduced at 4 l/min into the ESI source of either a Quattro LCZ, a tandem quadrupole instrument whose m/z range was specially extended to 8000, or a quadrupole time-of-flight instrument (Q-Tof) (Micromass UK Ltd., Wythenshawe, Manchester, United Kingdom) with a m/z range of 14,000.…”