Metal ion facilitated dissociation of heme from b-type heme proteins.
Addition of Ni(2+), Cu(2+), or Zn(2+) (10-40 equiv) to metMb in sodium bicarbonate buffer (25 degrees C) at alkaline pH (7.8-9.5) results in a time-dependent (2-6 h) change in the electronic absorption spectrum of the protein that is consistent with dissociation of the heme from the active site and that can be largely reversed by addition of EDTA. Similar treatment of cytochrome b(5), indoleamine 2,3-dioxygenase, and cytochrome P450(cam) (in the presence or absence of camphor) produces a similar spectroscopic response. Elution of metMb treated with Ni(2+) in this manner over an anion exchange column in buffer containing Ni(2+) affords apo-myoglobin without exposure to acidic pH or organic solvents as usually required. Bovine liver catalase, in which the heme groups are remote from the surface of the protein, and horseradish peroxidase, which has four disulfide bonds and just three histidyl residues, exhibit a much smaller spectroscopic response. We propose that formation of carbamino groups by reaction of bicarbonate with protein amino groups promotes both protein solubility and the interaction of the protein with metal ions, thereby avoiding precipitation while destabilizing the interaction of heme with the protein. From these observations, bicarbonate buffers may be of value in the study of nonmembrane proteins of limited solubility.
Mauk
Marcia R
MR
Department of Biochemistry and Molecular Biology, Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC V6T 1Z3 Canada.
Rosell
Federico I
FI
Mauk
A Grant
AG
D000818:Animals; D002164:Camphor / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry; D002417:Cattle; D003300:Copper / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D015786:Cytochromes b5 / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry*; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000737:chemistry; D008786:Metmyoglobin / Q000737:chemistry; D009532:Nickel / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry*
D002413:Cations, Divalent; D006420:Hemeproteins; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D008786:Metmyoglobin; D006418:Heme; D002164:Camphor; D003300:Copper; D009532:Nickel; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D015032:Zinc