International Journal of Biological Macromolecules xxx (2014) xxx–xxx
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International Journal of Biological Macromolecules
j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac
nvestigation on the chromium oxide interaction with solublehromatin and histone H1: A spectroscopic study
hatereh Khorsandi, Azra Rabbani-Chadegani ∗
epartment of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
r t i c l e i n f o
rticle history:eceived 3 March 2014eceived in revised form 12 June 2014ccepted 17 June 2014vailable online xxx
eywords:eavy metalshromium oxidehromatin
a b s t r a c t
Chromatin has been introduced as a tool for studying heavy metals action in nuclei. Chromium oxideis highly soluble and toxic with chronic exposure leading to mutagenesis and carcinogenesis. In thepresent study, for the first time, the binding affinity of chromium oxide to rat liver chromatin and histoneproteins was investigated. Reduction of chromatin absorbencies at 210 and 260 nm (hypochromicity) andfluorescence emission intensity upon metal binding represented quenching of the metal with chromatinchromophores. Binding isotherms demonstrated a positive cooperative binding pattern revealing higheraffinity of the metal to chromatin compared to DNA as confirmed by the binding constants. Meltingtemperature of chromatin was altered in a dose dependent manner and suggests partial removal ofhistones from the chromatin at metal concentrations higher than 15 �g/ml. Chromium oxide decreased
istone H1 the absorbance of histone H1 at 210 nm (hypochromicity) and fluorescence emission intensity revealedquenching of the metal with tyrosine residue located in the core domain of H1. Also the interaction ofchromium oxide with histone H1 increased its secondary structures. The results suggest toxic effect ofvery low concentrations of chromium oxide on chromatin and in this reaction both DNA and histonesare involved.
Organization of DNA in cells is achieved by folding and com-action into protein-DNA assemblies called chromatin [1]. Coreistone proteins in chromatin structure are composed of two copiesach of four proteins H2A, H2B, H3 and H4. Histone H1 is a veryysine-rich histone fraction of chromatin with a molecular weightf 21 KDa and binds to linker DNA between adjacent nucleosomeso facilitate compaction of DNA into higher order structures [2,3].t is now clear that chromatin plays an important role in mediat-ng the response to physiological and toxicological stimuli. This isighlighted by the fact that changes in the modification state andtructure of chromatin are among the fastest responses to toxicantxposure [4]. Toxicants may elicit their effects by targeting chro-
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atin structure directly which lead to mutation in components thategulate chromatin structure resulting human disease [5].
∗ Corresponding author at: Department of Biochemistry, Institute of Biochemistrynd Biophysics, University of Tehran, P.O. Box: 13145-1384, Tehran, Iran.el.: +98 21 66499422; fax: +98 2 66404680.
Chromium is one of the trace elements in the earth which existsin several oxidation states [6] and with the development of itsuses, the hazardous effects on human health has been defined [7].Also chromium (VI) compounds exert genotoxicity both in vivo andin vitro [8,9]. Chromium treatment of culture cells results in DNAstrand breaks and formation of stable chromium-DNA complexes[10]. Cross-linking of histone deacetylase1-DNA methyltransferase1 complexes to chromatin of Cyp1a1 promoter and the proteinsinvolved in chromatin structure is likely to change normal remod-eling and regulation of chromatin leading to inhibition of geneexpression [11,12]. Potential effect of chromium (VI) on epigenetichave also been reported demonstrating that the cells treated withCr (VI) increased di and tri methylation of lysine 9 in histone H3which in turn silences specific tumor suppressor genes [13].
We have recently reported the interaction of chromium oxidewith DNA [14] and shown that chromium oxide induces conforma-tional changes such as DNA condensation and perhaps cross-linkingbetween the strands. These functions happen through electrostaticinteraction, hydrogen bonding and mainly intercalation into basepairs of DNA. However, in the cell nucleus, DNA is compacted into
. Rabbani-Chadegani, Int. J. Biol. Macromol. (2014),
a complex structure built from DNA associated with basic proteinscalled histones introducing chromatin. Therefore chromatin, butnot naked DNA, is considered as a target for toxicants. In the presentstudy, we have focused on the interaction of chromium oxide
K. Khorsandi, A. Rabbani-Chadegani / International
ith chromatin in solution to obtain information about aspectsf chromium toxicity at chromatin level. The results demonstratehat chromium oxide binds to chromatin with higher affinity thano DNA and histone proteins play an important role in chromiumxide toxicity.
. Materials and methods
.1. Chemicals
Chromium oxide [CrO3, Cr (VI)] (Merck) was dissolved in de-onized double distilled water as a stock solution (2 mg/ml) andtored at 4 ◦C. Calf thymus DNA (Sigma) was dissolved in deionizedouble distilled water dialyzed against 10 mM Tris–HCl (pH 7.3)nd stored at 4 ◦C for a month without any turbidity and absorbancehanges. DNA concentration was determined spectrophotometri-ally using extinction coefficient of 20 cm2 mg−1 at 260 nm.
.2. Preparation of chromatin and histone H1
Soluble chromatin was prepared as described [15] and itsoncentration determined using an extinction coefficient of600 cm2 mg−1 at 260 nm.
Histone H1 was extracted by 0.25 N HCl as described by Johns16] and further purified. The protein was then dissolved in 10 mMris–HCl (pH 7.2) and after pH adjustment; the solution stored at20 ◦C and used within a month.
.3. UV/vis spectroscopy
Soluble chromatin [50 �g/ml] and histone H1 [100 �g/ml] werencubated individually in the absence and presence of various con-entrations of chromium oxide in 10 mM Tris–HCl (pH 7.2), for5 min at 23 ◦C. Various concentrations of the metal was incubatedlong with the treated samples under the same experimental con-ition and used for comparison. Metal treated and the controlsere then subjected to spectroscopic analysis using shimadzu UV-
60 spectrophotometer (Japan), equipped with quartz cuvettes atulti � system. The wavelength of 350 nm was selected as �max
f chromium oxide and 260 and 210 nm which is related to theavelength of DNA and protein. Difference spectra were drawn for
hromatin–chromium oxide complexes between 190 and 300 nmgainst various concentrations of the metal in the same buffer.
.4. Fluorescence measurement
The measurement was carried out on a Carry Eclipse flu-rescence spectrophotometer equipped with a thermostaticallyontrolled cell holder at ambient temperature. The monochro-atic slits were set at 5 nm and 10 nm for excitation and emission
espectively to reduce the intensity of the signal depending on thexperiment. All samples were made in 10 mM Tris–HCl (pH 7.2) at3 ◦C and quartz fluorescence cell of 1 cm path length was used.hromatin (20 �g/ml) and histone H1 (25 �g/ml) were incubated
ndividually in the absence and presence of various concentrationsf chromium oxide for 45 min at room temperature in the dark.or chromatin samples, the spectra were recorded between 500nd 540 nm after excitation at 258 nm and also at 290–390 nmfter excitation at 278 nm. For histone H1, a wavelength range of90–390 nm after excitation at 278 nm was employed. Chromiumxide, chromatin and histone H1 were prepared individually inhe same buffer and their emission spectra recorded in the same
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ondition and used as a reference. The (Io − I/Io × 100) values, forach sample, was estimated and normalized with respect to theuorescence of samples in the absence of the metal in which Iond I are fluorescence emission intensity before and after addition
PRESSl of Biological Macromolecules xxx (2014) xxx–xxx
of the metal, respectively. Stern–Volmer constant (Ksq) is used toevaluate the fluorescence quenching efficacy [17].
2.5. Circular dichroism analysis
Circular dichroism (CD) was performed using CD spectrometermodel 215 (AVIV instruments Inc., USA). Near-UV CD spectrum ofchromatin (100 �g/ml) was recorded in the range of 200–310 nmin 10 mM Tris–HCl (pH 7.2) in the absence and presence of variousconcentrations of chromium oxide. The far-UV CD spectrum of his-tone H1 [200 �g/ml] was recorded in the range of 190–260 nm inthe same condition with a spectral resolution of 1 nm. Quartz cellwith a path length of 10 mm was used and all measurements werecarried out at 23 ◦C. The results were expressed as molar ellipticity[�], in deg × cm2 × dmol−1.
2.6. Thermal denaturation profiles
Thermal denaturation profiles of chromatin in the absence andpresence of chromium oxide was obtained using stopped quartzcuvettes on a carry 100 Bio-spectrophotometer. Absorbance mon-itored at 260 nm with a temperature-scanning rate of 1 ◦C/min.The slope of melting profiles at temperature T was obtained usingdh(T)/dT = h(T + 1) − h(T − 1)/2 [18].
2.7. Equilibrium dialysis
Soluble chromatin (100 �g/ml) and DNA (100 �g/ml) were pre-pared in 10 mM Tris–HCl (pH 7.2) and dialyzed against the samebuffer containing serial concentrations of chromium oxide usingSpectrum Laboratories (USA) dialysis tubing at room temperature.Equilibrium was achieved within 72 h at 23 ◦C. Total metal concen-tration (Ct) and the concentration of free metal (Cf) in the dialysatewas measured directly from the absorbance at 350 nm (�max ofthe metal) before and after dialysis using extinction coefficient of2173 M−1 cm−1. The amount of bound metal (Cb) was obtained fromCb = Ct − Cf. Binding parameters were determined from the plot ofr/Cf versus r according to Scatchard method [19], where r is the ratioof bound metal to total base pair concentration and Cf is the amountof free metal. In this presentation, n (the apparent number of bind-ing sites) is intercept of the linear region of binding curve with thehorizontal axis. Also K (apparent binding constant) corresponds tothe negative value of the slope of the curve. Hill coefficient (nH) wasdetermined from the slope of ln(r/nr) versus ln Cf according to Hillequation [18].
2.8. Statistical analysis
Statistical analysis was performed with Student’s t-test (twotailed). P < 0.05 was considered as statistically significant. Resultsare expressed as means ± SD with n denoting the number of exper-iments.
3. Results
3.1. UV/vis spectroscopy represents binding of chromium oxide tochromatin
Soluble chromatin was incubated in the absence and presenceof various concentrations of chromium oxide and UV absorbancechanges estimated by subtracting the absorbance of free metalfrom that of chromatin–metal complex. Fig. 1A shows absorbance
. Rabbani-Chadegani, Int. J. Biol. Macromol. (2014),
changes upon increasing chromium oxide concentration. As is seenaddition of chromium oxide to chromatin solution resulted in sig-nificant absorbance decrease at 210 nm whereas at 260 nm theabsorbance is slightly increased and then remains unchanged up to
K. Khorsandi, A. Rabbani-Chadegani / International Journal of Biological Macromolecules xxx (2014) xxx–xxx 3
F ence o( oxideo P < 0.0
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ig. 1. (A) UV absorbance changes of chromatin (100 �g/ml) in the absence and prespH 7.2). (B) Difference spectra drawn against the same concentration of chromiumf chromium oxide respectively. The results are expressed as the mean ± SD (n = 3,
00 �g/ml of the metal. At high concentration of chromium oxidebsorbance is decreased (P < 0.005). Fig. 1B demonstrates metalffect on chromatin spectrum and shows considerable absorbanceecrease at 210 nm which accompanies with red shift.
.2. Quenching of chromium oxide with chromatin chromophores
To study the binding of chromium oxide to chromatin, vari-us amounts of the metal were added to a constant concentrationf chromatin and the fluorescence emission intensity recordedetween 500 and 540 nm after excitation at 258 nm. Fig. 2A rep-esents fluorescence emission spectra of chromatin in the absencend presence of chromium oxide. As is seen, the fluorescence emis-ion intensity of chromatin is decreased as metal concentration isncreased. To found out whether chromium oxide has any effectn protein chromophores in chromatin structure, emission inten-ity recorded between 290 and 380 nm after excitation at 278 nm.s is shown in Fig. 2B, chromatin in the absence of chromiumxide exhibits maximum fluorescence intensity at 335 nm corre-ponding to aromatic amino acids. It should be mentioned thatistone proteins lack tryptophan but contain low contents of tyro-ine and phenylalanine residues. Addition of chromium oxide tohe chromatin solution reduces the fluorescence intensity of chro-
atin with slight blue shift in the emission maxima (Imax) as metaloncentration is increased.
Fig. 2C shows the binding affinity of chromium oxide to chro-atin derived from data of Fig. 2A and B. As is seen, for DNA
excitation at 258 nm) the binding affinity increases up to 15 �g/mlf the metal and then levels off. In the case of proteins regionexcitation at 278 nm) the binding affinity is similar to DNA pat-ern but more metal values is needed to obtain saturation state.ig. 2D displays Stern–Volmer plot showing quenching effect ofhromium oxide on chromatin chromophores. According to thelassical Stern–Volmer equation Io/I = 1 + Ksq [chromium oxide] asentioned in the method section. The Ksq value is obtained as a
inear plot with a value of 0.15 M−1 for chromium oxide.
.3. Chromium oxide alters secondary structure and thermaltability of chromatin
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Circular dichroism and thermal denaturation have emergeds the most sensitive techniques to probe and provide informa-ion on the interaction of biomolecules with metals. To determinehether the binding of chromium oxide to chromatin has any
f chromium oxide at 260 ( ) and 210 nm ( ). Reaction buffer was 10 mM Tris–HCl at each point in the same buffer. Spectra 1–6 are 0, 10, 25, 50, 100, and 200 �g/ml
05).
effect on secondary structure, CD spectra were carried out inthe presence and absence of various concentrations of chromiumoxide. As shown in Fig. 3A chromatin in the absence of themetal provides two extremes, a positive extreme at 275 nm whichcorresponds to DNA and negative extremes between 208 and220 nm related to the protein part of chromatin. No extremeat 245 nm is observed for chromatin possibly due to mask-ing of DNA molecule with histone proteins. In the presenceof chromium oxide the ellipticity is significantly decreased at275 nm upon addition of the metal. At low concentration ofthe metal, ellipticity at 208 and 220 nm becomes more nega-tive, whereas, at higher concentration of chromium oxide theellipticity is increased (becomes positive) and shifts to higher wave-length.
Derivative thermal denaturation profile of chromatin in theabsence and presence of chromium oxide was examined (at260 nm), and the result is shown in Fig. 3B. The melting tempera-ture (Tm) is defined where the half of the total base pairs is unboundand determined as transition of melting curve. In the absence ofchromium oxide, chromatin exhibits a typical thermal profile withTm at 85 ◦C. Upon addition of chromium oxide Tm is increased to89–90 ◦C when 15 �g/ml of the metal is used and accompanies withhypochromicity. Whereas at higher concentration (>15 �g/ml) Tmis considerably shifter to lower values (84 ◦C). The Tm changes isshown in the table in the inset of Fig. 3B.
3.4. Cooperative binding of chromium oxide to chromatin andDNA
To compare the binding parameters of the interaction ofchromium oxide with chromatin and DNA, binding isotherms wereestimated from equilibrium dialysis experiment and the result isshown in Fig. 4. Scatchard plot of the binding of chromium oxide tosoluble chromatin exhibits a cooperative binding pattern as illus-trated by a positive slope observed in the lower regions of thebinding isotherm. The curve reaches a maximum at a value of r = 0.5and decreases in the slope is observed at higher r-values. DNAalso shows a positive cooperative binding pattern with a maxi-mum r-value of 0.75. Drawing r versus Cf, as shown in Fig. 4B,clearly demonstrates a sigmoid curve, that is, as Cf increases,
. Rabbani-Chadegani, Int. J. Biol. Macromol. (2014),
r raises rapidly and then levels off, indicating that the systemapproaches to equilibrium or saturation. The binding of chromiumoxide to chromatin and DNA represents a binding constant ofKa = 2.1 × 102 M−1 and Ka = 0.99 × 102 M−1 respectively. Drawing
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ARTICLE IN PRESSG ModelBIOMAC 4419 1–7
4 K. Khorsandi, A. Rabbani-Chadegani / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
Fig. 2. Fluorescence emission spectra of chromatin (50 �g/ml) in the absence and presence of different concentrations of chromium oxide. Excitation was at 258 nm (A) andat 278 nm (B). All samples were prepared in reaction buffer (10 mM Tris–HCl, pH 7.2) and the incubation time after the metal addition was 45 min. Spectra 2–6 are 5, 10, 15,20 and 40 �g/ml of chromium oxide, respectively. The abbreviation (a.u.) is for arbitrary units. (C) Denotes Io − I/Io values for chromatin interaction with chromium oxide. Ioand I are fluorescence intensity before and after addition of the metal, respectively. (D) Stern–Volmer plot of fluorescence quenching of chromatin by chromium oxide. (D)Number of individual experiments was 3.
Fig. 3. CD spectra of chromatin (A) in 10 mM Tris–HCl at pH 7.2 in the absence and presence of various concentrations of chromium oxide. Each spectrum was obtained at23 ◦C with a 10 mm path length cell. 1–6 are 0, 10, 25, 50, 100 and 200 �g/ml of chromium oxide, respectively (n = 3). (B) Derivative thermal denaturation profile of chromatinin the absence and presence of chromium oxide. Curves 1–9 are 0, 2.5, 5, 10, 15, 25, 50, 100 and 200 �g/ml of chromium oxide, respectively. Reaction buffer was 10 mMTris–HCl (pH 7.3) and profile was drawn at 260 nm. Table in the inset represents Tm values and the results are expressed as the mean ± SD.
K. Khorsandi, A. Rabbani-Chadegani / International Journal of Biological Macromolecules xxx (2014) xxx–xxx 5
F romiu(
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ig. 4. Scatchard plots (A) and saturation state (B) obtained for the interaction of chpH 7.2) for 72 h at 23 ◦C. The results are means of three individual experiments.
n (r/n − r) against ln Cf gives a straight line with a slope of nH
Hill coefficient) with a value of 1.8 (chromatin) and 1.6 (DNA)onfirming positive cooperative binding of the metal. Using thequation of �G◦ = −RTln Ka, total macroscopic negative free energyalues of −3.2 and −2.7 K cal/mol were obtained for the binding ofhromium oxide to chromatin and DNA respectively.
.5. Affinity of chromium oxide to histone H1 in solution
The observations that chromium oxide shows different affin-ty to DNA compared to chromatin encouraged us to determine
hether histone proteins play a role in this binding process. Inhromatin structure, the four core histones produce an octamerhich is covered with DNA making them less accessible to the
nvironment [2] whereas, histone H1 as a linker-DNA binding pro-ein is almost exposed to the environment. Therefore in this study,he interaction of chromium oxide with histone H1 was exam-ned in the same experimental condition used for the chromatino obtain more information about the mode of chromium oxidection at the chromatin level. As we know, histone proteins areasic proteins with very low content of aromatic amino acids,.g. histone H1 contains only one tyrosine and one phenylalanineocated in the core/globular head of its structure. The purified his-one H1 was incubated in the absence and presence of variousoncentrations of chromium oxide and then absorbance changesonitored at 210 nm. As is seen in Fig. 5A, absorbance of histone H1
t 210 nm is considerably decreased is the presence of chromiumxide (P < 0.005).
The fluorescent emission spectra were drawn between90–390 nm with excitation at 278 nm. As is seen in Fig. 5B, his-one H1 free in solution, exhibits fluorescence emission intensityn the position corresponding to tyrosine with a maximum intensityt 305–308 nm [20]. Addition of chromium oxide reduces fluores-ence intensity of histone H1 without any red shift in the emissionaxima (Imax). The fluorescence emission intensities were normal-
zed to protein concentration, Io − I/Io estimated and the result ishown in Fig. 5C. As is shown, binding affinity of chromium oxideo histone H1 is increased upon increasing metal concentration andhen reaches to saturation state. Fig. 5D displays Stern–Volmer plotor the quenching effect of chromium oxide on histone H1 with Ksq
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alue of 0.24 M−1.To determine whether the binding of chromium oxide to his-
one H1 has any effect on its secondary structure, CD spectrumf histone H1 was drawn in the absence and presence of various
m oxide with soluble-chromatin ( ) and DNA ( ) carried out in 10 mM Tris–HCl
concentrations of chromium oxide. As is seen in Fig. 6, histoneH1 in the absence of the metal exhibits one negative extreme at198 nm because histone H1 is essentially random-coil in low ionicstrength buffers as has been reported previously [21]. Upon addi-tion of chromium oxide to histone solution, ellipticity is decreasedor becomes more negative.
4. Discussion
Chromium is widely used in many industries such as chro-mate manufacturing, chrome plating, ferrochrome production, andstainless steel welding. Recent studies have revealed that expo-sure to chromium compounds lead to various types of cancers [6].Until recently, alteration of gene expression mediated by DNA dam-age is considered the key mechanism underlying genotoxic andcarcinogenic activity of chromium; however, several studies havehighlighted the potential epigenetic effects of chromium oxidewhich contribute to its toxicity [8,9,13]. The present study describesan attempt to characterize the interaction of chromium oxide withchromatin and histone H1 in solution to elucidate genotoxicity ofthis metal at chromatin level.
From the results it is suggested that the interaction of chromiumoxide with chromatin is different from that of naked DNA [14].In chromatin, DNA is covered by histone proteins lowering thebinding potential of the metal to DNA. This is clearly shown bycomparing the binding parameters. Estimation of binding param-eters suggest a cooperative binding pattern for the binding ofchromium oxide to both chromatin and DNA however the affin-ity to chromatin is higher as the binding constants confirmed it(Ka value of 2.1 × 102 M−1 and 0.99 × 102 M−1 for chromatin andDNA respectively). We have obtained higher binding constant forDNA compared to the report of Arakawa et al. [22] which can beattributed to different experimental condition used. Since the Gibbsfree energy changes show negative values, the binding process isspontaneous (exergonic).
UV absorbance and fluorescence spectroscopy analysis clearlydemonstrate binding affinity of chromium oxide to chromatin.Absorbance at 260 nm is less affected compared to 210 nm indi-cating participation of histone protein (maximum absorbance at210 nm) as well as sugar-phosphate backbone of DNA in the
. Rabbani-Chadegani, Int. J. Biol. Macromol. (2014),
chromium oxide–chromatin complex formation. Reduction of flu-orescence emission intensity of chromatin upon exposure tochromium oxide reveals quenching of chromatin chromosphereswith the metal and also participation of histone proteins in
6 K. Khorsandi, A. Rabbani-Chadegani / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
Fig. 5. The effect of chromium oxide on UV absorption changes of histone H1 (A), fluorescence emission spectra with excitation at 278 nm (B). Spectra 1–6 are 0, 5, 10, 15,2 itraryI ly. (DR n ± SD
cbfcroototoihdotcohMip
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0 and 40 �g/ml of chromium oxide, respectively. The abbreviation (a.u.) is for arbo and I are fluorescence intensity before and after the addition of metal respectiveeaction buffer was 10 mM Tris–HCl (pH 7.2). The results are expressed as the mea
hromium oxide-chromatin interaction. Therefore with no doubtoth DNA and histone proteins can be introduced as a main targetor the chromium binding in chromatin structure. Compaction ofhromatin in the presence of chromium oxide is observed by theesults obtained from thermal denaturation profiles. Occurrencef hypochromicity and Tm changes of chromatin upon chromiumxide binding is dose dependent, thus, at low concentration ofhe metal, chromatin structure is stabilized (Tm is increased). Inther words the binding of the metal precedes chromatin struc-ure into compaction a hypochromicity is a sign of surface bindingf the metal. Whereas, at higher concentrations (<15 �g/ml) Tms decreased suggesting destabilization of chromatin. Significantypochromicity represents that no single stranded DNA is pro-uced therefore reduction in Tm values at higher concentrationsf the metal can be attributed to the release (partially) of his-ones from chromatin upon chromium oxide binding which shiftshromatin Tm into lower degrees. Reduction of CD ellipticityf chromatin at 275 nm clearly demonstrates alteration of DNAelicity and stacking of the bases upon chromium oxide binding.oreover, ellipticity of chromatin at 222 nm becomes more pos-
tive and shifted into higher wavelengths (red shift) which implyossible loosening of histones from chromatin.
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This finding reveals evidence about the binding affinity ofhromium oxide to histone proteins. As we know, core histonesH2A, H2B, H3 and H4) make an octamer which is covered by DNAn the structure of core particle, hence they are less accessible to the
units. (C) Denotes Io − I/Io values for histone H1 interaction with chromium oxide.) Stern–Volmer plot of fluorescence quenching of histone H1 by chromium oxide.
(n = 3, P < 0.005).
environment, whereas, histone H1 as a linker DNA binding proteinis more exposed and affected by the ligand binding to chromatin[23,24]. The structure of H1 consists of three main domains; N-terminal and C-terminal domains are rich in basic amino acids andhave random coil structure (especially C-terminal which is highlypositively charged). The central domain is hydrophobic with a glob-ular structure. Also, histone H1 contains only one-tyrosine andone-phenylalanine residues as a flour but lack tryptophan [25].Quenching of chromium oxide with tyrosine residue as revealed byfluorescence emission intensity reduction at 305–310 nm suggeststhat the globular part of histone H1 is the site of metal quench-ing. In dilute buffers or under physiological condition used in theseexperiments, and absence of any reducing agent, chromium oxide ismostly in the form of CrO4
2− which can easily bind to basic (lysine)through ionic interaction, however, H-bind via donor and accep-tors of H1 and the metal cannot be ignored. This is in agreementwith the report of Levina et al. that by using vibration electron X-ray absorption spectroscopy and performing the interaction of Cr(VI) with poly l-lysine and histones in solution have shown thatas lysine residues of H3 is the site of chromium binding. Also theyconclude that Cr (VI) binds to newly synthesized histone in cyto-plasm and then Cr (VI)-histone complex inters the nuclei and binds
. Rabbani-Chadegani, Int. J. Biol. Macromol. (2014),
to DNA [26].Moreover, binding of chromium oxide to histone H1 reduce
the absorbance at 210 nm and CD ellipticity becomes more nega-tive indicating that the protein in the presence of chromium oxide
K. Khorsandi, A. Rabbani-Chadegani / International Journa
Fig. 6. CD spectra of histone H1 in 10 mM Tris–HCl at pH 7.2 in the absence and pres-ence of chromium oxide. Each spectrum was obtained at 23 ◦C with a 10 mm pathlN
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[[[26] A. Levina, H.H. Harris, P.A. Lay, J. Biol. Inorg. Chem. 11 (2006) 225–234.
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ength cell. 1–6 are 0, 10, 25, 50, 100 and 200 �g/ml of chromium oxide, respectively.umber of individual experiments was 3.
hows more compact structure compared to the protein in thebsence of the metal which confirms the strong ionic binding ofhe metal to the lysine residues.
Apart from our previous report on the strong interaction ofhromium oxide with DNA [14], there is a report suggesting that CrVI) has very low or no affinity to DNA [27]. Their conclusion is that,n the cell, under physiological conditions Cr (VI) can be reducedy reductants (glutathione, ascorbic acid, H2O2, etc.) to produceeactive intermediates [Cr (V), Cr (IV)] and ultimately Cr III whichnteracts with DNA [28]. But in our experimental condition in whicho reductant and Cr (III) exists chromium (VI) oxide itself binds tohromatin upon high exposure of the cells, in which chromium eas-ly inters the nuclei and binds by binding to histones in chromatintructure inhibiting chromatin normal function. It is confirmed byhe work of Nowicka et al. showing that in cancer cells, Cr (VI) butot Cr (III) species decrease the affinity if anticancer drugs to DNA29].
From the results it is finally concluded that chromium oxide
Please cite this article in press as: K. Khorsandi, Ahttp://dx.doi.org/10.1016/j.ijbiomac.2014.06.018
ffects chromatin structure by binding to both DNA and histone.stimation of EC50 value clearly demonstrates that histone pro-eins play a fundamental role in the chromium oxide binding tohromatin (EC50 for chromatin and histone H1 was 13 �g/ml and
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PRESSl of Biological Macromolecules xxx (2014) xxx–xxx 7
8 �g/ml respectively). Also quenching value (Ksq) confirmed it(Ksq) for histone and chromatin was 0.24 M−1 and 0.15 M−1 respec-tively. Toxic effect of chromium oxide on chromatin may affect thebiological function of chromatin especially transcription and repli-cation in which precedes the cellular progression into apoptosis andnecrosis. Although our experiments on bone marrow cells confirmsthe toxic effect of chromium oxide at the cellular level (unpublishedresults) more work is needed to elucidate the real mechanism ofchromium oxide action on chromatin components especially, thehistones.
Acknowledgment
The authors would like to acknowledge the University of Tehran
for financial support to this work under grant # 6401017/6/18.
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