Interaction of hemin with placental glutathione transferase.
To verify a possible involvement of glutathione transferase pi in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase pi was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 microM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase pi in the hemin transport.
Caccuri
A M
AM
Dipartimento di Biologia, Università di Roma, Tor Vergata, Italy.
Aceto
A
A
Piemonte
F
F
Di Ilio
C
C
Rosato
N
N
Federici
G
G
D001663:Bilirubin / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D002942:Circular Dichroism; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D006418:Heme; D006427:Hemin; D005982:Glutathione Transferase; D005978:Glutathione; D001663:Bilirubin