The dissociation of the extracellular hemoglobin of Lumbricus terrestris at acid pH and its reassociation at neutral pH. A new model of its quaternary structure.

Abstract

Lumbricus terrestris HbOz and HbCO dissociated below pH 5.0; a time-dependent alteration to the met form occurred at pH < 5 and pH < 4.5, respectively.The extent of dissociation was unaffected by alkaline earth cations but was decreased by an increase in ionic strength.HbOz and HbCO exposed to pH 4.0-4.8were centrifuged to obtain the undissociated pellet (Pl) and dissociated supernatant (Sl) fractions.S1 was reassociated at pH 7.0 by dialysis against various buffers and then centrifuged to obtain the reassociated pellet (P2) and unreassociated supernatant (S2) fractions.Reassociation was possible only if S1 was dialyzed against water prior to return to neutral pH; otherwise precipitation occurred starting at about pH 5.3.The extent of reassociation varied from about 40 to 80%, was usually higher for HbCO than HbOz, and was unaffected by an increase in ionic strength or by Ca(I1).Gel filtration of P2 on Sephacryl S-300 at neutral pH gave one peak IaR, eluting at a slightly greater volume than the native Hb; S1 and S2 gave in addition, three peaks, Ib (200 kDa), I1 (65 kDa), and I11 (18 kDa).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that P2 was slightly deficient in subunit M relative to the Hb, that Ib was deficient in subunits D l and D2 and that I1 and I11 consisted of subunits D l + D2 + T and subunit M, respectively.Scanning transmission electron microscopy of P2 showed that it was smaller than the native hemoglobin: 25 nm in diameter and 16 nm in height, instead of 30 % 20 nm.Comparison of the results of the dissociations of Lumbricus H b at alkaline pH (Kapp, 0. H.,