Please wait a minute...

Frontiers in Biology

Front. Biol.    2016, Vol. 11 Issue (1) : 32-42     DOI: 10.1007/s11515-016-1388-0
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
Download: PDF(926 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

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 Authors: 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.
E-mail this article
E-mail Alert
Articles by authors
Pijush Basak
Tanay Debnath
Rajat Banerjee
Maitree Bhattacharyya
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
1 Arias-Moreno X, Abian O, Vega S, Sancho J, Velazquez-Campoy A (2011). Protein-cation interactions: structural and thermodynamic aspects. Curr Protein Pept Sci, 12(4): 325–338
doi: 10.2174/138920311795906664 pmid: 21401523
2 Bardhan M, Mandal G, Ganguly T J (2009). Steady state, Time resolved and Circular dichroism spectroscopic studies to reveal the nature of interactions of zinc oxide nanoparticles with transport protein Bovine Serum Albumin and to monitor the possible protein conformational changes. Appl Phys (Berl), 106(3): 34701–34705
doi: 10.1063/1.3190483
3 Basak P, Bhattacharyya M (2013). Intrinsic tryptophan fluorescence and related energy transfer in Leghemoglobin isolated from Arachis hypogeal. Turkish. J Biochem, 38: 9–13
4 Basak P, Pattanayak R, Bhattacharyya M (2015). Transition metal induced conformational change of heme proteins. Spectrosc Lett, 48(5): 324–330
doi: 10.1080/00387010.2014.881380
5 Berezin M Y, Achilefu S (2010). Fluorescence lifetime measurements and biological imaging. Chem Rev, 110(5): 2641–2684
doi: 10.1021/cr900343z pmid: 20356094
6 Berlett B S, Levine R L, Stadtman E R (2000). Use of isosbestic point wavelength shifts to estimate the fraction of a precursor that is converted to a given product. Anal Biochem, 287(2): 329–333
doi: 10.1006/abio.2000.4876 pmid: 11112281
7 Cantor C R, Schimmel P R (1984). Biophysical Chemistry Part II:Techniques for the Study of Biological Structure and Function. New York: W.H.Freeman and Company, 386–398
8 Das P, Mallik A, Halder B, Chakraborty A, Chattopadhyay N (2006). Effect of nanocavity confinement on the rotational relaxation dynamics: 3-acetyl-4-oxo-6,7-dihydro- 12 H indolo- [ 2, 3- a ] quinolizine in micelles. J Chem Phys, 125(4): 044516
doi: 10.1063/1.2219751
9 Dickerson R E, Timkovich R (1975). The Enzymes, (P. Boyer Ed.). New York: Academic Press
10 Faergeman N J, Sigurskjold B W, Kragelund B B, Andersen K V, Knudsen J (1996). Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry. Biochemistry, 35(45): 14118–14126
doi: 10.1021/bi960545z pmid: 8916897
11 Giovannetti R, Uddin J (2012). The Use of Spectrophotometry UV-Vis for the Study of Porphyrins, InTech, 95–96, ISBN: 978–953–51–0664–7
12 Gourion-Arsiquaud S, Chevance S, Bouyer P, Garnier L, Montillet J L, Bondon A, Berthomieu C (2005). Identification of a Cd2+- and Zn2+-binding site in cytochrome c using FTIR coupled to an ATR microdialysis setup and NMR spectroscopy. Biochemistry, 44(24): 8652–8663
doi: 10.1021/bi050322l pmid: 15952772
13 Harbury H A, Loach P A (1960). Oxidation-linked proton functions in heme octa- and undecapeptides from mammalian cytochrome c. J Biol Chem, 235: 3640–3645
pmid: 13711455
14 Heringa J, Argos P (1991). Side-chain clusters in protein structures and their role in protein folding. J Mol Biol, 220(1): 151–171
doi: 10.1016/0022-2836(91)90388-M pmid: 2067014
15 Hua Y J, Liua Y I, Zhanga L X, Zhaoa R M, Qua S S (2005). Studies of interaction between colchicine and bovine serum albumin by fluorescence quenching method. J Mol Struct, 750(1-3): 174–178
doi: 10.1016/j.molstruc.2005.04.032
16 Kadish K M, Smith K M, Guilard R (2010). Handbook of Porphyrin Science. World Scientific Publishing: Singapore
17 Kahn K, Bruice T C (2003). Comparison of reaction energetics and leaving group interactions during the enzyme-catalyzed and uncatalyzed displacement of chloride from haloalkanes. J Phys Chem B, 107(28): 6876–6685
doi: 10.1021/jp022407r
18 Kang J, Liu Y, Xie M X, Li S, Jiang M, Wang Y D (2004). Interactions of human serum albumin with chlorogenic acid and ferulic acid. Biochim Biophys Acta, 1674(2): 205–214
doi: 10.1016/j.bbagen.2004.06.021 pmid: 15374625
19 Lakowicz J R (2006). Principles of Fluorescence Spectroscopy, 3rd ed. New York: Plenum Press, 277–285
20 Lehrer S S (1971). Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry, 10(17): 3254–3263
doi: 10.1021/bi00793a015 pmid: 5119250
21 Liao M S, Watts J D, Huang M J (2006). DFT/TDDFT study of lanthanide(III) mono- and bisporphyrin complexes. J Phys Chem A, 110(48): 13089–13098
doi: 10.1021/jp0632236 pmid: 17134170
22 Marchon J C, Mashiko T, Reed C A (1982). Electron-Transport and Oxygen Utilization. (C.H.O. Ed.) North Holland, New York: Elsevier
23 Mata L, Sanchez L, Calvo M (1997). Interaction of mercury with human and bovine milk proteins. Biosci Biotechnol Biochem, 61(10): 1641–1645
doi: 10.1271/bbb.61.1641 pmid: 9362112
24 Mátyuss L, Szöllosi J, Jenei A (2006). Steady-state fluorescence quenching applications for studying protein structure and dynamics. J Photochem Photobiol B, 83(3): 223–236
doi: 10.1016/j.jphotobiol.2005.12.017 pmid: 16488620
25 Muller P (1994). Glossary of terms used in physical organic chemistry. Pure Appl Chem, 66(5): 1077
doi: 10.1351/pac199466051077
26 Murayama M (1958). Titrable sulphahydryl groups of Hemoglobin C and fetal Hemoglobin at 0° and 38°. J Biol Chem, 230: 163–168
pmid: 13502384
27 Murayama M (1959). On the nature of the interaction between binding sites for heavy metals (mercapto-mercapto interactions) in normal human hemoglobin. J Biol Chem, 234: 3158–3162
pmid: 14425330
28 Murphy C B, Zhang Y, Troxler T, Ferry V, Martin J J, Jones W E (2004). Probing Förster and Dexter energy-transfer mechanisms in fluorescent conjugated polymer chemosensors. J Phys Chem, 108(5): 1537–1543
doi: 10.1021/jp0301406
29 Nada T, Terazima M (2003). A novel method for study of protein folding kinetics by monitoring diffusion coefficient in time domain. Biophys J, 85(3): 1876–1881
doi: 10.1016/S0006-3495(03)74615-3 pmid: 12944300
30 Ordway G A, Garry D J (2004). Myoglobin: an essential hemoprotein in striated muscle. J Exp Biol, 207(Pt 20): 3441–3446
doi: 10.1242/jeb.01172 pmid: 15339940
31 Pauling L (1960). The Nature of the Chemical Bond (3rd Edn.), Ithaca, NY: Cornell University Press
32 Pinto M R, Schanze K S (2004). Amplified fluorescence sensing of protease activity with conjugated polyelectrolytes. Proc Natl Acad Sci USA, 101(20): 7505–7510
doi: 10.1073/pnas.0402280101 pmid: 15136727
33 Raphael A L, Gray H B (1991). Semisynthesis of axial-ligand (position 80) mutants of cytochrome c. J Am Chem Soc, 113(3): 1038–1040
doi: 10.1021/ja00003a045
34 Rispens T, Lakemond C M M, Derksen N I, Aalberse R C (2008). Detection of conformational changes in immunoglobulin G using isothermal titration calorimetry with low-molecular-weight probes. Anal Biochem, 380(2): 303–309
doi: 10.1016/j.ab.2008.06.001 pmid: 18577365
35 Ross P D, Subramanian S (1981). Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry, 20(11): 3096–3102
doi: 10.1021/bi00514a017 pmid: 7248271
36 Samanta U, Pal D, Chakrabarti P (2000). Environment of tryptophan side chains in proteins. Proteins, 38(3): 288–300
doi: 10.1002/(SICI)1097-0134(20000215)38:3<288::AID-PROT5>3.0.CO;2-7 pmid: 10713989
37 Takeda K, Hachiya K (2002). Interaction of Protein with Ionic Surfactant, Part I: Marcel Dekker; New York
38 Wachter R M, Elsliger M A, Kallio K, Hanson G T, Remington S J (1998). Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein. Structure, 6(10): 1267–1277
doi: 10.1016/S0969-2126(98)00127-0 pmid: 9782051
39 Walker V E, Castillo N, Matta C F, Boyd R J. (2010). The Effect of multiplicity on the size of iron(II) and the structure of iron(ii) porphyrins. J Phys Chem A, 114:10315–10319
40 Wang J, Guo D, Yuan X (2006). Influence of copper on the interaction between cytochrome c and sulfite in vitro. J Biochem Mol Toxicol, 20(5): 255–258
doi: 10.1002/jbt.20143 pmid: 17009250
41 Wuthrich R Q (1985). Amino acid sequence, haem iron coordination geometry and functional properties of mitochondrial and bacterial c-type cytochromes. Rev Biophys, 18(02): 111–134
doi: 10.1017/S0033583500005151
42 Yan Y, Marriott G (2003). Analysis of protein interactions using fluorescence technologies. Curr Opin Chem Biol, 7(5): 635–640
doi: 10.1016/j.cbpa.2003.08.017 pmid: 14580569
43 Zaidi N, Ahmad E, Rehan M, Rabbani G, Ajmal M R, Zaidi Y, Subbarao N, Khan R H (2013). A comprehensive insight into binding of hippuric acid to human serum albumin: A study to uncover its impaired elimination through hemodialysis. J Phys Chem B, 117: 2595–2604
doi: 10.1021/jp3069877 pmid: 23438181
44 Zhang Y Z, Xiang X, Mei P, Dai J, Zhang L L, Liu Y (2009). Spectroscopic studies on the interaction of Congo Red with bovine serum albumin. Spectrochim Acta A Mol Biomol Spectrosc, 72(4): 907–914
doi: 10.1016/j.saa.2008.12.007 pmid: 19155189
Full text