Interaction of zinc protoporphyrin with intact oxyhemoglobin.
In erythropoietic protoporphyria and lead poisoning, free protoporphyrin (PPIX) and zinc protoporphyrin (ZPP), respectively, accumulate in erythrocytes. That PPIX and ZPP bind to human hemoglobin A (Hb4) is established, but the site of binding is still a matter of controversy. We investigated the interaction of ZPP with intact, tetrameric oxy Hb4, using batch microcalorimetry, front-face fluorometry, absorption difference spectroscopy, oxygen equilibrium studies, and isoelectric focusing (IEF). In the presence of oxy Hb4 (pH 7.35, 0.05 M phosphate), the fluorescence emission maximum (excitation at 420 nm) of ZPP immediately shifts from 587 nm (ZPP alone) to 594 nm, as expected when binding to protein. The fluorescence intensity increases with time and is correlated with the ZPP:Hb4 mole ratio. A slow, time-dependent reaction is also observed with microcalorimetry: the rate of heat of reaction exhibits both a fast and a slow component. The heats of reaction range from -2.1 to -14.8 mcal depending upon the ZPP:Hb4 ratio of 4:1 (0.4 mM:0.1 mM) to 38:1 (3.8 mM:0.1 mM), respectively, and are typical of weak, noncovalent protein-ligand interactions. The optical difference spectra are a function of the ZPP:Hb4 molar ratio and also exhibit a slow increase in intensity over time. No time-dependent optical difference spectra are observed with ZPP or with Hb4 alone. The oxygen affinity of Hb4 in the presence of ZPP decreases with increasing mole ratio. During IEF, all ZPP separates from Hb4, consistent with a weak, noncovalent interaction at a non-heme pocket site. We conclude that ZPP binds to intact, tetrameric hemoglobin at non-heme pocket sites in a nonspecific, weak, noncovalent interaction.
Hirsch
R E
RE
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
Lin
M J
MJ
Park
C M
CM
D002151:Calorimetry / Q000379:methods; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010108:Oxyhemoglobins / Q000378:metabolism*; D011166:Porphyrins / Q000378:metabolism*; D011485:Protein Binding; D011524:Protoporphyrins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry / Q000379:methods
D046911:Macromolecular Substances; D010108:Oxyhemoglobins; D011166:Porphyrins; D011524:Protoporphyrins; C017803:zinc protoporphyrin