Hemin binding

Xu, X., Holt, S. C. and Kolodrubetz, C. (2001). Cloning and expression of two novel hemin binding protein genes from Treponema denticola, Infect. Immun., 69, 4465 1472. 

The following example describing hemin binding to human a i-acid glycoprotein illustrates the latter two paragraphs. 

Binding studies are performed between ai-acid glycoprotein and hemin, a co-factor that binds to the hydrophobic pocket of hemoproteins. If a i-acid glycoprotein contains a hydrophobic pocket, hemin will bind to this pocket with a well defined stoichiometry. Also, if TNS and hemin bind to the same binding site and if the affinity of hemin to ai-acid glycoprotein is more important than that of TNS, binding of hemin to a protein-TNS complex will remove the TNS from its binding site. 

Addition of hemin to a solution of a fixed amount of ai-acid glycoprotein - TNS complex induces a complete abolishment of TNS fluorescence ( iex, 320 nm and 420 nm) by 1 mol hemin / mol ai-acid glycoprotein (Fig. 6.10). Thus, TNS and hemin bind to the same site on a i-acid glycoprotein. 

Since, hemin is known to bind to the hydrophobic pockets of hemoproteins, one should expect that hemin binds to the only hydrophobic site of a i-acid glycoprotein, its pocket. 

The stoichiometry of ai-acid glycoprotein - hemin complex (1 1) indicates that the hemin binds to a specific site. Displacement of TNS by hemin gives the nature of this site a hydrophobic one. Therefore, hemin does not bind to the surface of ai-acid glycoprotein, a hydrophilic area. It binds to a hydrophobic domain of the pocket of the protein inducing a decrease of the fluorescence intensity of Trp residues of the protein via a Forster energy transfer mechanism. 

Energy transfer studies between Trp residues and TNS and calcofluor white gave values for Ro equal to 28 A (Albani et al. 1995) and 18 A (Albani, 2003), respectively. The very close values of Ro for Trp -> TNS and Trp -> hemin energy transfer is also an indication of the fact that TNS and hemin binds to the same hydrophobic site within ap acid glycoprotein pocket. 

Thus, the decrease of the fluorescence intensity of Trp residues beyond the stoichiometry concentration is the result of a non specific quenching mechanism by aggregated hemin binding nonspecifically to the protein (Albani, 1985). 

In chapter 6 paragraph 3, we have described experiments showing that hemin binds to ap acid glycoprotein in a hydrophobic pocket. These experiments are a direct proof of the presence of a pocket in a - acid glycoprotein. In the absence of crystallographic data on the protein, the results described in paragraphs 11a and 6.3 are the only direct experiments performed up to now that put into evidence the presence of a pocket in ap acid glycoprotein. 

While activation of HSFl and HSF2 results in the increased expression of HS genes, recent studies indicate that there are distinct differences between these transcription factors. As noted previously, HSFl is the heat-inducible form of the HS factors, whereas HSF2 is activated by hemin (Sistonen et al., 1992). This observation has indicated a possible role of HSF2 in cellular differentiation. It also indicates that the activation domains of the two transcription factors are quite different. Presumably the DNA binding properties of the two transcription factors are similar, but recent experiments indicate otherwise (Kroger and Morimoto,... 

Cyanide binding to the hydroxo complex of ferric hemes has been extensively studied in micellar solutions [15-17, 22, 34]. The binding of cyanide is found to be highly dependent on the pH and is maximum at pH 9.6. Optical spectral studies show a distinct isosbestic point at 412 nm for the formation of bis-cyano hemin in SDS from the hydroxo heme. The overall equilibrium is ... 

Iron(n) is known to decompose hydrogen and dialkyl peroxides to free radicals by reductive cleavage of the 0—0 bond and early investigations established the parasite s sensitivity to these species. When treated with radiolabelled C-artemisinin, the hemin-hemozoin fraction of the lysed malaria-infected erythrocytes was shown to contain a radiolabel, though the mechanism of incorporation is not clear. Meshnick and coworkers demonstrated that uninfected cells did not contain radiolabelled proteins whereas six radiolabelled proteins were isolated from cells infected with the Plasmodium falciparum (P. falciparum) strain of the parasite. It was suspected that one of the alkylated proteins was the Histidine Rich Protein (HRP) that was known to bind multiple heme monomers and therefore thought to be instrumental to the parasite s detoxification process. Moreover, iron chelators were found to inhibit the lethal effects of peroxides on the parasite. ... 

NOTE Ferroprotoporphyrin is the term used for reduced heme (mol. wt. 616 Windholz et al., 1976). Hematin (mol. wt. 633) is one of several terms used for the oxidized or ferric heme. Other terms include ferriporphyrin hydroxide or ferriheme hydroxide, due to the binding of a hydroxyl to the ferric heme iron. Hornsey (1956) used the term acid hematin (mol. wt. 652) to describe the heme oxidation product in acidified acetone solutions. The more common term hemin is used in this unit. It is also known as chlorohemin, due to binding of a chloride ion to the ferric heme iron. 

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