Electrospray ionization mass spectrometric determination of the molecular mass of the similar to 200-kDa globin dodecamer subassemblies in hexagonal bilayer hemoglobins

Hexagonal bilayer hemoglobins (Hbs) are similar to 3.6-MDa complexes of sim ilar to 17-kDa globin chains and 24-32-kDa, nonglobin linker chains in a si milar to 2:1 mass ratio found in annelids and related species, Studies of t he dissociation and reassembly of Lumbricus terrestris Hb have provided amp le evidence for the presence of a similar to 200-kDa linker-free subassembl y consisting of monomer (RI) and disulfide-bonded trimer (T) subunits, Elec trospray ionization mass spectrometry (ESI-MS) of the subassemblies obtaine d by gel filtration of partially dissociated L, terrestris and Arenicola ma rina Hbs showed the presence of noncovalent complexes of IM and T subunits with masses in the 213.3-215.4 and 204.6-205.6 kDa ranges, respectively. Th e observed mass of the L. terrestris subassembly decreased linearly with an increase in de-clustering voltage from similar to 215,400 Ha at 60 V to si milar to 213,300 Ha at 200 V, In contrast, the mass of the A. marina comple x decreased linearly from 60 to 120 V and reached an asymptote at similar t o 204,600 Ha (180-200 V). The decrease in mass was probably due to the prog ressive removal of complexed water and alkali metal cations, ESI-MS at an a cidic pH showed both subassemblies to consist of only M and T subunits, and the experimental masses demonstrated them to have the composition M,T,. Be cause there are three isoforms of M and four isoforms of T in Lumbricus and two isoforms of M and 5 isoforms of T in Arenicola, the masses of the M,T, subassemblies are not unique. A random assembly model was used to calculat e the mass distributions of the subassemblies, using the known ESI-MS masse s and relative intensities of the M and T subunit isforms, The expected mas s of randomly assembled subassemblies was 213,436 Ha for Lumbricus Hb and 2 04,342 Ha for Arenicola Hb, in good agreement with the experimental values.