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Frontiers in Biology

ISSN 1674-7984

ISSN 1674-7992(Online)

CN 11-5892/Q

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Front. Biol.    2016, Vol. 11 Issue (1) : 32-42    https://doi.org/10.1007/s11515-016-1388-0
RESEARCH ARTICLE
Selective binding of divalent cations toward heme proteins
Pijush Basak1,Tanay Debnath2,Rajat Banerjee3,Maitree Bhattacharyya1,*()
1. Department of Biochemistry, University of Calcutta, 35, Ballygunge Circular Road, Kolkata-700019, India
2. Department of Chemistry, Indian Institute of Chemistry Kanpur, Kanpur 208016, U. P. India
3. Department of Biotechnology, University of Calcutta,35, Ballygunge Circular Road, Kolkata-700019, India
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Abstract

Potential toxicity of transition metals like Hg, Cu and Cd are well known and their affinity toward proteins is of great concern. This work explores the selective nature of interactions of Cu2+, Hg2+ and Cd2+ with the heme proteins leghemoglobin, myoglobin and cytochrome C. The binding profiles were analyzed using absorbance spectrum and steady-state fluorescence spectroscopy. Thermodynamic parameters like enthalpy, entropy and free energy changes were derived by isothermal calorimetry and consequent binding parameters were compared for these heme proteins. Free energy (DG) values revealed Cu2+ binding toward myoglobin and leghemoglobin to be specific and facile in contrast to weak binding for Hg2+ or Cd2+ . Time correlated single photon counting indicated significant alteration in excited state lifetimes for metal complexed myoglobin and leghemoglobin suggesting bimolecular collisions to be involved. Interestingly, none of these cations showed significant affinity for cytochrome c pointing that, presence of conserved sequences or heme group is not the only criteria for cation binding toward heme proteins, but the microenvironment of the residues or a specific folding pattern may be responsible for these differential conjugation profile. Binding of these cations may modulate the conformation and functions of these biologically important proteins.
Keywords heme proteins      divalent cations      fluorescence quenching      isothermal calorimetry      time correlated single photon counting (TCSPC)     
Corresponding Author(s): Maitree Bhattacharyya   
Just Accepted Date: 22 February 2016   Online First Date: 16 March 2016    Issue Date: 22 March 2016
 Cite this article:   
Pijush Basak,Tanay Debnath,Rajat Banerjee, et al. Selective binding of divalent cations toward heme proteins[J]. Front. Biol., 2016, 11(1): 32-42.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-016-1388-0
https://academic.hep.com.cn/fib/EN/Y2016/V11/I1/32
Fig.1  UV-Vis absorption spectra of myoglobin in presence of increasing concentration (0 to 30 μM) of (A) CuCl2 (B) HgCl2 and (C) CdCl2 .
Fig.2  Stern-Volmer plot (A) and modified Stern-Volmer plot (B) for fluorescence quenching of leghemoglobin by CuCl2 and HgCl2.
Protein Metal SV equation R2 Ksv (106 mol-1) Modified SV log[(F0-F)/F] = logKa + nlog[Q] R2 Binding constant(Ka)(mol-1) No. of sites ofbinding Free energy(DG)(kJ·mol-1)
Mb CuCl2 Y= 0.0169X + 1 0.989 0.169 Y= 0.918X+ 3.851 0.994 7.1 × 103 1 -21.97
HgCl2 Y= 0.0041X + 1.075 0.983 0.041 Y= 0.521X+ 1.740 0.975 0.55 × 102 0.52 -9.93
Lb CuCl2 Y= 0.0160X + 1.075 0.929 0.16 Y= 0.914X+ 3.385 0.993 7.07 × 103 0.91 -21.95
HgCl2 Y= 0.0011X + 1.030 0.949 0.011 Y= 0.521X+ 1.740 0.969 0.56 × 102 0.52 -9.97
Tab.1  Binding constant and relevant parameters by SV and modified S-V plot in fluorescence quenching
Protein Ligand Binding constant(mol-1) Enthalpy change (?H)(cal/mol) Entropy change (?S)(cal/(mol·deg K)) Free energy change(?G) (kJ·mol-1) Remarks
Myoglobin CuCl2 2.89 × 104 -5.3 × 104 -157 -25.44 ExothermicEnthalpy driven
HgCl2CdCl2 981.37× 104 5.65 × 105-3.875× 104 1.9 × 103-0.119 × 103 -11.36-0.32×104 EndothermicEntropy and enthalpy drivenExothermicEnthalpy driven
Leghemoglobin CuCl2 2.39 × 104 6.65 × 104 243 -24.98 EndothermicEntropy and enthalpy driven
HgCl2CdCl2 602.26 × 104 5.71 × 1065.8 × 104 1.9 × 1040.267 × 103 -10.14-2.10×104 EndothermicEntropy and enthalpy drivenEndothermicEntropy and enthalpy driven
Tab.2  Thermodynamic parameters of Protein-divalent cation interaction derived by ITC method
Fig.3  Isothermal titration calorimetric profile for the Lb-divalent interaction with samples at pH 7.0 (0.1M phosphate buffer) at 22°C. The raw data indicates addition of Lb (A) CuCl2, (B) HgCl2, (C) CdCl2.
Fig.4  Time-resolved fluorescence decays of native Lb and Lb– metal complex respectively with excitation wavelength of 295 nm. Concentration of Lb: 3 mM, and each metal concentration: 30 mM.
Fig.5  Time-resolved fluorescence decay of native Mb and Mb – metal complex respectively with excitation wavelength of 295 nm. Concentration of Mb: 3 mM, and each metal concentration: 30 mM.
Fig.6  Time-resolved fluorescence decays of native Cyt C and Cyt C – metal complex respectively with excitation wavelength of 295 nm. Concentration of Cyt C: 3 mM, and each metal concentration: 30 mM.
Protein α1 t1 α2 t2 α3 t3 Average (t) c2
Leghemoglobin Native 0.12 0.30 0.36 2.0 0.52 5.43 2.57 1.21
CuCl2 0.10 0.13 0.21 0.82 0.69 2.90 1.28 1.40
HgCl2 0.14 0.60 0.27 1.47 0.59 3.55 1.87 1.13
CdCl2 0.16 0.40 0.32 1.73 0.52 3.90 2.01 1.11
Myoglobin Native 0.06 0.08 0.24 1.91 0.70 4.99 2.32 1.27
CuCl2 0.05 0.06 0.15 1.33 0.80 3.38 1.59 1.30
HgCl2 0.07 0.09 0.210 1.80 0.72 3.73 1.87 1.20
CdCl2 0.07 0.09 0.20 1.40 0.73 5.03 2.17 1.33
Cytochrome c Native 0.16 0.70 0.28 1.99 0.56 3.98 2.12 1.08
CuCl2 0.13 0.25 0.29 1.60 0.58 3.27 1.70 1.30
HgCl2 0.10 0.22 0.26 1.40 0.64 3.97 1.86 1.04
CdCl2 0.09 0.14 0.180 1.80 0.73 4.14 2.02 1.23
Tab.3  Fluorescence life time decay profile of protein-divalent cation interaction by TCSPC method
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