The role of the dodecamer subunit in the dissociation and reassembly of the hexagonal bilayer structure of Lumbricus terrestris hemoglobin.
The dissociation of the approximately 3500-kDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris upon exposure to Gdm salts, urea and the heteropolytungstates [SiW11O39]8- (SiW), [NaSb9W21O86]18- (SbW) and [BaAs4W40O140]27- (AsW) at neutral pH was followed by gel filtration, SDS-polyacrylamide gel electrophoresis, and scanning transmission electron microscopy. Elution curves were fitted to sums of exponentially modified gaussians to represent the peaks due to undissociated oxyHb, D (approximately 200 kDa), T+L (approximately 50 kDa), and M (approximately 25 kDa) (T = disulfide-bonded trimer of chains a c, M = chain d, and L = linker chains). OxyHb dissociation decreased in the order Gdm*SCN > Gdm.Cl > urea > Gdm.OAc and AsW > SbW > SiW. Scanning transmission electron microscopy mass mapping of D showed approximately 10-nm particles with masses of approximately 200 kDa, suggesting them to be dodecamers (a+b+c)3d3. OxyHb dissociations in urea and Gdm.Cl and at alkaline pH could be fitted only as sums of 3 exponentials. The time course of D was bell-shaped, indicating it was an intermediate. Dissociations in SiW and upon conversion to metHb showed only two phases. The kinetic heterogeneity may be due to oxyHb structural heterogeneity. Formation of D was spontaneous during HBL reassembly, which was minimal (</= 10%) without Group IIA cations. During reassembly, maximal (approximately 60%) at 10 mM cation, D occurs at constant levels (approximately 15%), implying the dodecamer to be an intermediate.
Sharma
P K
PK
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Kuchumov
A R
AR
Chottard
G
G
Martin
P D
PD
Wall
J S
JS
Vinogradov
S N
SN
D000818:Animals; D000845:Annelida / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D046911:Macromolecular Substances; D008706:Methemoglobin; D017348:Microscopy, Electron, Scanning Transmission; D010108:Oxyhemoglobins / Q000737:chemistry*; D011485:Protein Binding; D011489:Protein Denaturation; D014508:Urea / Q000737:chemistry
D006146:Guanidines; D046911:Macromolecular Substances; D010108:Oxyhemoglobins; D014508:Urea; D008706:Methemoglobin; D019791:Guanidine; D002118:Calcium