QUATERNARY STRUCTURE OF THE EXTRACELLULAR HEMOGLOBIN OF THE LUGWORM ARENICOLA-MARINA - A MULTI-ANGLE-LASER-LIGHT-SCATTERING AND ELECTROSPRAY-IONIZATION-MASS-SPECTROMETRY ANALYSIS

To elucidate the quaternary structure of the extracellular haemoglobin (Hb) of the marine polychaete Arenicola marina (lugworm) it was subje cted to multi-angle laser-light scattering (MALLS) and to electrospray -ionisation mass spectrometry (ESI-MS). It was also subjected to SDS/P AGE analysis for comparative purposes. MALLS analysis gave a molecular mass of 3648 +/- 24 kDa and a gyration radius of 11.3 +/- 1.7 nm. Max imum entropy analysis of the multiply charged electrospray spectra of the native, dehaemed, reduced and carbamidomethylated Hb forms, provid ed its complete polypeptide chain and subunit composition. We found, i n the reduced condition, eight globin chains of molecular masses 15 95 2.5 Da (a1), 15 974.8 Da (a2), 15 920.9 Da (b1), 16 020.1 Da (b2), 16 036.2 Da (b3), 16 664.8 Da (c), 16 983.2 Da (d1), 17 033.1 Da (d2) and two linker chains L1, 25 174.1 Da, and L2, 26 829.7 Da. In the native Hb, chains b, c, d occur as five disulphide-bonded trimer subunits T with masses of 49 560.4 Da (T1), 49 613.9 Da (T2), 49 658.6 Da (T3), 4 9 706.8 Da (T4), 49 724.5 Da (T5). Linker chains L1 and L2 occur as on e disulphide-bonded homodimer 2L1 (DI) of 50 323.1 Da and one disulphi de-bonded heterodimer L1-L2 (D2) of 51 981.5 Da. Polypeptide chains a and d possess one free cysteine residue and chains d possess an unusua l total of five cysteine residues. Semi-quantitative analysis of ESI-M S data allowed us to propose the following model for the one-twelfth p rotomer: [(3a1)(3a2)(2)T] (T corresponding to either T3, T4 or T5). Fr om electron micrograph data T1 and T2 are probably located at the cent re of the molecule as mentioned in previous studies. The Hb would thus be composed of 198 polypeptide chains with 156 globin chains and 42 l inker chains, each twelfth being in contact with 3.5 linker subunits, providing a total mass of 3682 kDa including haems in agreement with t he experimental molecular mass determined by MALLS. From ESI-MS relati ve intensities and the model proposed above, the globin/linker ratio g ave 0.71:0.29 and 0.73:0.27, respectively. The estimation of haem cont ent by pyridine haemochromogen and by cyanmethaemoglobin (HiCN) method s also support the globin chain number provided by ESI-MS.