Kinetic and spectroscopic studies of haemoglobin and myoglobin from Urechis caupo. Distal residue effects.
Seven components of the tetrameric haemoglobin (Hbu) from Urechis caupo were separated by preparative isoelectric focusing and characterized by their absorption spectra and pI values. The helix content and Soret delta epsilon values are reported for several of the components. Temperature-jump O2-binding kinetics of the major components of Hbu show biphasic behaviour, with the majority species having kon = 1.57 x 10(9) mol-1.s-1 and koff = 3.32 x 10(4) s-1. The Fourier-transform i.r. spectrum of pooled Hbu(II)-CO displays a stretching frequency of 1942 cm-1. E.s.r. of Hbu(II)-NO demonstrates evidence of proximal strain similar to that encountered in T-state human haemoglobin. CO-driven reduction of U. caupo methaemoglobin, Hbu(III) and U. caupo metmyoglobin [Mbu(III)] shows much higher rates relative to haemoglobins and myoglobins known to possess a distal histidine residue. Nitrosyl auto-reduction kinetics of Hbu(III)-NO and Mbu(III)-NO are examined. The equilibrium binding constants of several ligands are reported for both Hbu and Mbu, and together with the above kinetic data suggest differences in haem pocket environments between Hbu and Mbu. Reaction of Hbu with 2-chloromercuri-4,6-dinitrophenol demonstrates the presence of one reactive thiol group per globin chain. lambda max. values and the respective molar absorption coefficients for selected ligand-bound states are reported for the major component of Hbu and for Mbu. The majority haem orientation in U. caupo haemoglobin is identical with that of human haemoglobin.
DiFeo
T J
TJ
Department of Chemistry, Drexel University, Philadelphia, PA 19104.
Addison
A W
AW
Stephanos
J J
JJ
D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004606:Elephants; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007448:Invertebrates / Q000378:metabolism*; D007525:Isoelectric Focusing; D007700:Kinetics; D008706:Methemoglobin / Q000378:metabolism; D008786:Metmyoglobin / Q000378:metabolism; D009211:Myoglobin / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013438:Sulfhydryl Compounds / Q000378:metabolism
D006454:Hemoglobins; D009211:Myoglobin; D013438:Sulfhydryl Compounds; D008786:Metmyoglobin; D009569:Nitric Oxide; D002248:Carbon Monoxide; D008706:Methemoglobin; D010100:Oxygen