314 - Electrochemical oxidation of nucleic acids and proteins at graphite electrode. Qualitative aspects

BioeZecZrochenzisrrusfryand J_

EZectroanaZ_

Elsevier

Chenz.

Sequoia

Bioener,oefics 116

S.A.,

(IgSo)

Lausanne

7 (rgSo)

69-52

69-52 -

Printed

in Italy

314 - Ele&roch&ical Oxidation of Nucleic Acids and Proteins at Graphite Ekctrode- Qualitative Aspects * by Institute

VIKTOR of

BR~BEC

Biophysics.

Czechoslovak

Academy

of

Sciences.

612 65 Brno.

Cze-

choslovakia Manuscript

received

September

9th rg7g

The electrochemical oxidation of DNAs differing in the content of guauine plus cytosine (G-l-C) was investigated at a pyrolytic graphite electrode by means of differential pulse (DP) voltammetryAt pH 6.4 all samples of DNA studied yielded a peak G on DP voltammo,wms corresponding to the osidation of the guanine residues, and a peak A corresponding to the osidation of the adenine residues. The potentials of the peaks G and A were not influenced by the G+C content in DNA and differed by o_aS V_ It was found that the ratio of the heights of the peaks A and G was identical with great accuracy to the ratio of the contents (adenine+thyxnine) and (G+C)_ This fact was exploited for developing a new method for the determination of the G+C content in DNA.

The electrochemical oxidation of proteins at a spectroscopic graphite electrode impregnated with paraffin was (WISGE) was studied by means of linear sweep, cyclic, and DP \-oltammetry- It was found that proteins were electrochemically oxidizable at the WISGE. They yielded a faradaic peak on v-oltammograms in the vicinity of 0_7-0-S V in a neutral medium_ The voltammetric study of proteins, poly-(aminoacids). peptides of known aminoacid composition and free aminoacids revealed that the irreversible electroo_xidation of tyrosine (and, contingently, of tryptophan) residues is responsible for the appearance of the protein peak at the WISGE. It is suggested that DP voltammetry at a graphite electrode might become another electrochemical method suitable for studies of conformational changes of proteins, and in particnhrr of those not containing cystine or cysteine (e.g. histones). ImmductIon Investigation chemical analysis

of nucleic acids and proteins using methods of electrohas already brought several significant findings con-

* Invited istry,

3-S

lecture at the 5th International September 1979, Weimar (D-D-R.).

o3oz-_lg$3/So/oo6g-ooSz

Q

Elsevier

Sequoia

Symposium

S-1.

on

Bioelectrochem-

70

Brabec

cerning the properties of these biopolymers not only in the interphase, but also in bulk solution [x-7]_ Up to now, mostly polarographic methods, ie_ methods exploiting DALE. as the indicator electrode E3-6. S]. have been used for the electrochemical analysis of nucleic acids and proteinsThe properties of nucleic acids when they interact with eIectricaIIy charged surface were also investigated using H_XD_E_ [I. 2.4. S]_ The use of mercury electrodes in investigation of biopo_lymers enabled the study of electroreduction of adenine and cytosine resrdues in nucleic acids [I. 2.5.6, Sj. and of cystine residues in proteins, or of non-protein electroactive groups in conjugated proteins [6. S]_ BRDICRA'S catalytic reaction of proteins in ammoniacal solution of cobalt salts [g, IO] belongs also to the known manifestations of the electrochemical activity of proteins at the mercury electrode_ The potential limit of mercury electrodes at pH 7 varies in the range from about 0 to -IS (vs_ S-C-E.) depending on the composition of the background electrolyte_ They are thus especially applicable for the investigation of the electroreduction of organic substances_ The study of the electrooxidation of organic substances is made possible above all by the use of some stationary electrodes, whose potential Iimit permits measurements even at relatively highly positive potentials [II]_ We have demonstrated recently that nucIeic acids are electrochemically oxidizable at graphite electrodes [73_ Measurements by means of differential pulse (DP) voItammetry at the pyrolytic graphite eiectrode (PGE) showed [7. IZ] that nucleic acids yield two well separated osidation peaks on DP voltammograms in a broad region of pH and ionic strength of the medium (e-g_, in neutral media in the vicinity of o-g \; and 1.2 V)_ The more negatrve peak G corresponds to the electrooxidation of guanine residues, whrle the more positive peak A corresponds to the eIectroosidation of the adenine residues_ In the \-icinity of pH 7 the electrooxidation of guanine and adenine residues at the PGE is markedly suppressed in natrve DNA as compared with thermally denatured DNA [IZ~_ This is because denatured DNA is relativeiy flexible and. when adsorbed on a ,wphite electrode surface, conforms to the contours of the rough electrode surface ; thus more adenine and guanine residues are accessible to the electrode process and relatively large voltammetric peak currents are observed_ On the other hand, native DNA has a more rigid structure and cannot conform so readily to the contours of the electrode surface when adsorbed on a graphite electrodeHence fewer adenine and guanine residues are accessible for interaction with the eIectrode and smailer differential puise voltammetric peaks are observed_ The fact that the DNA osidizability at a graphite electrode depends on its conformation was exploited in a study of themral and acid denaturation of DNA [131 and of the denaturation induced by ionizing and ultra\-iolet -radiations [x4]_ The electrochemical osidation of proteins at stationary electrodes has not yet been described. Besrdes a brief review of the most important results of the electroosidation of nucleic acids published up to now, in the present paper we also endeavour to show, how the electrochemical osidizabilitv of DNA at ,qphite electrodes was influenced by the content of guau&e+

Electrooridation

of

DX_X

and

Proteins

i*

cytosine (G+C)_ Finally this paper is also aimed at demonstrating that even proteins are electrochemically oxidizable at graphite electrodes, a fact which opens up a possibility of developing a new technique for protein analysis. Experimental Materials

Samples of DNA of Micrococcus ktezrs (72 Tk G+C) and of calf thymus (42 o/o G+C) were isolated as described in previous papers [rj,x6j, DNA samples of EscLrerichia cdi (50 y. G-i-C). BaciZZm certws (33 7; G+C). bacteriophage l-2 (34 y. G+C), and mouse lymphosarcoma cells (40 y/o G+C) were made available to me by courtesy of Drs E. La16Sov_<, V_ ICLEIXWXCHTER, >I_ VORLI~KO~~. and J_ KEPRTOY~, respecti\-ely_ DN_A isolated from chicken blood (42 y. G+-C) was purchased from P EASAL (Hungary) _ The RN.1 content in the DNA samples, estimated by means of orcinoI [x71. was less than I Ok, the protein content, as determined by the method of LOWRY et al_ [IS] did not exceed o-5 yb, and the content of denatured DKA. estimated pulse-polarographicaily [$, did not exceed I Oh_ Before using DNAs for voltammetric measurements they were dialyzed against sodium phosphate of ionic strength o-01 and pH 7 for 4s hours [7]_ The denaturation of DNA was performed by heating DNX (in a concentration which was twice the one in which the voltammetric measurements were carried out) in sodium phosphate of ionic strength 0.01 and pH 7 at IOO 0C for IO min. followed by quick cooling in an ice bath_ Lysozyme (of hen egg white) was obtained from NUTRITIOSAL BIOCHEJIICALS (Ohio), ribonuclease (of bovine pancreas) and bovine serum albumin from FE_asaL (Hungary). insulin (of bovine pancreas) and all aminoacids from CALBIOCHEJI (California) ; poly-L-tyrosine and copolymer poly(L-tyrosine,L-tryptophan) (the tyrosine : tryptophan ratio was 4:r). a product of XILES LABORATORIES (Israel), were kindlydonated by Dr_ V_ KLEISWXCHTER ; giutathion (reduced form) was obtained from KOCH-LIGHT Laboratories (Engiand). apamin from SEW-A (Heidelberg)_ Histone HI was a kind gift of Dr_ AI_ SK_~LKA_ Urea and chemicals used for preparation of background electrolyte solution (all of analytical grade) were obtain<-d from LACHEX~ (Bmo). The PGE and paraffin i-_-:x impregnated spectroscopic graphite electrode (WISGE) were made and used in the same way as described earlier [r2]_ The PGE, WISGE No_ I. and WISGE Xo_ 2 had geometric areas of ca 3 mm=, 7 mm=, and 30 mm4, respectively_

The voltammograms of DNXs at the PGE xere obtained in the same way as described in our previous papers [7, ra-14]_ Linear sweep

(LS) and DP voltammograms of proteins, peptides, and aminoacids were obtained in a 2 cm” capacity thermostatted cell_ A three-electrode system was used, including the WISGE, a Pt counter_eIectrode of appreciable area, and a saturated mercurylmercurous sulphate electrode ; however. all potentials given in this paper were re-calculated against S-C-E_ LS and cyclic voltammetric measurements at the WISGE were carried out with a GWP 673 %rltimode Polarographic Analyzer (GDR)DP voltammetric measurements of proteins at the WISGE were performed with a prototype of a pulse polarograph PA I (LABORATORY ISSTRUXESTS, Prague)_ _ DP voltammograms of proteins were obtained with pulse amplitude of 50 mV and sweep rate of 3-33 mV s-r. The current sampling for DP voltammetry at the WISGE was set with the drop time control of the PA I set at 1.0 s_ LS and DP voltammograms of proteins were recorded on an EXDIM 620.02 recorder (GDR). Cyclic voltammograms were recorded with an OG2-21 Speicheroszilloskop, VEB XESSELEKTROXIK Berlin (GDR) in the X-J- mode_ The basic procedures for DP voltammetry of DNA at the PGE have been described previously [7. 12-r& The procedures for the voitammetry of proteins, peptides, and aminoacids at the WISGE were only slightly different from those used for the voltammetry of DNA_ Once the WISGE was inserted into the ‘tested solution contained in an electrochemical cell, it was allowed to stand for IO s without applied potential_ Then, if not stated otherwise, the initial potential (0.2 V) was applied for the next 120 s, after which time the voltammetric sweep was started; for the first 60 s of the applied initial potential a magnetic stirrer rotated at the bottom of the electrochemical cell at a speed of cu_ 300 rj_m_ The macroscale electrolysis at controlled potential was performed with the aid of GWP 673 three-electrode system_ The working electrode was represented by a bundle of seven WISGE’s, each of them having a geometric area of 30 mm9 We electrolyzed S cm3 of ribonuclease solution at the concentration of 50 l&cm” in BRITTOS-ROBIXSOX buffer, pH 74 _A magnetic stirrer rotated at the bottom of the cell at a speed of 300 r$_m_ during the electrolysis_ Solutions of DNA, proteins, peptides and aminoacids were prepared for \-oltammetric measurements in the following way: a solution of suitable buffer was pipetted dropwise into an equal volume of a solution of DNA, protein, peptide or aminoacid to obtain the desired composition and pH @H was measured after mixing) Poly-(aminoacids) and histone fraction Hi, which are difficult to dissolve in aqueous solutions, were dissolved directly in the background electrolyteAll voltammetric measurements mere carried out with the voltammetric cell at 23 oC_ All potentials reported in this paper are given VCYSZCS the S-C-E_ at 25 oC_

Electrooridation

of DNA

and Proteins

Deter4ninatiota of the conte4zt of guani4te+cytosirte 44zean.sof voZtamntetry at graplr-ite electrodes

ire

73

DN_4

sa44zples b,v

The content of G+C in DNA is an important factor infiuencing the properties of DNA to a considerable extent. Therefore great attention has been devoted to the methods of its determination_ Considering the fact that the methods of electro-chemical analysis have found possibilities of extensive application in the investigation of nucleic acids, it is not surprising that they were used successfully even for the determination of the G+C content in DNA_ One of these methods is based on the measure of the temperature at which a cathodic indentation on oscillopolarograms dU/dt =f(U) disappears in the course of the thermal deThe advantage of this method was naturation of a DNA sample [Ig]_ its rapidity (e-g_ in comparison with spectrophotometric measurements), the disadvantage was low accuracy and the requirement of highly purified DNA samples_ Another electrochemical method for the determination of the content of G+C in DNA is also based on the oscillopolarographic

a

0.6

0.8

1.0

1.2

Fig_ I. Differential pulse voltamtiograms of Tz bacteriophage DN_% at the PGE in o-2 M sodium acetate, pH 6.4 (u) 300 &cms native DNA. (b) 300 &cm’ denatured DNAThe value x and y were taken to represent the heights of peaks G and A. respectively_

activity of DNA [zo]_ It was found that an anodic indentation of thermally denatured DNA observed on osciliopolarograms dU/dt = f(U) depended on the G-)-C content of DNA. However, the determination of the content of G+C in an unknown DNA sample by the latter method required the construction of a suitable calibration curve. The list of DXAs used. their content of guaninefcytosine Table I_ the ratio of the heights of difierential puke-voltammetric peaks A DSXs at the PGE.

Source

of

DKh

‘3’0G-i-C

Bacillics ccreus Bacteriophage Mouse

Iymphosarcoma

cells Chicken CaIf

Tz

blood

thymus

Eschericl~ia coti Micrococcus

1zrffms

(XtT)j(G+C)

(G-W). and and G (K) of

(h’)*,yP

(1.1*&W

33 34

Z-03 r-94

2.00 2 -00

2.00 I.92

40

I-50

I-54

I_52

41 *’

1.3s r_3S

1.2s

=-3i r-35

50

r-32

1.00

7"

1.00 0.40

o-39

1.00

o-&o

+ The indexes mat and dmat are related DSX. pH

respectively.

in concentration

to native DS.1 and thermally denatured of 300 pg:/crn3 and in 0.~ M sodium acetate.

64

As already mentioned, nucleic acids yield _ two peaks on DP volt1 ._ ammograms (Fig. I) ; the more negative peak ti corresponds to tne oxidation of guanine residues at the PGE. the more positive peak A to the oxidation of adenine residues at the PGE_ If DP voltammograrns mere recorded for native and denatured DNA samples from various sources (Table I) at pH 6.4, it was found that the potentials of the peaks G and A were independent of the G+C content in DNA. The ratio of the heights of the peaks A and G (K) was for each DNA sample identical with a great accuracy to the ratio of the base contents, (adeninefthymine): (guaninef This fact thus makes possible a cytosine) (A-i-T) : (G+C) (Table I)_ very rapid and direct determination of the G+C content in DNA by means of DP voItammetry at the PGE according to the relationship

G+C

(%)

=

iota T

I

(1)

The accuracy of the determination of the G+C content in DNA by this method depends on the the accuracy of the measurement of the peaks A and G_ As shown in Fig. xb, the thermally denatured DNA yields

Electrooxidation

of DNA

and Proteins

75

substantially better developed peaks A and G. Accordingly, in order to increase the accuracy of the determination, it is useful to denature thermally the DNA sampie_ The heights of both peaks A and G increase with increasing DNA concentration and tend to a limiting value [7, IZ]. Therefore it is important to analyze the sample of an unknown DNA in However. considering the limiting the highest possible concentration. character of the concentration dependence of the heights of the peaks A and G. increasing the concentration above ca 300 pgjcm3 does not Iead to any significant improvement of the measurability of the peaks A and G. The peaks A and G of DNA having molecular weight of 2 x 107 were measurable even at concentration of 50 vg DNA per cm3_ For DNAs of molecular weight of ca I x xo6 and lower, this limit markedly decreased with decreasing molecular weight. Voltammetric measurements at the PGE can be carried out in a microcell of the volume of 0.2-o.3 cm3, so that 10-20 p,u DNA is sufficient for the determination, depending on the molecular weight of the sample_ A study of the influence of the molecular weight of DNA on the determination of the G+C content in DNA by means of DP voltammetry at the PGE [21] showed that the relationship (I) can be used at least in the range of molecular The determination was also not influenced weights I x 10% - z x IO’. by the presence of proteins, RNA, and polysaccharides in concentrations not exceeding ca IO O/O of the DNA content in the samples. However, the determinations were markedly influenced by the presence of monomeric purine bases and their derivatives, short oligonucleotides containing purine bases, as well as by the presence of inorganic cations of certain salts capable of shifting the anodic potential limit of the graphite electrodes to more negative values (e-g_ of chloride ions)_ Therefore it is necessary to dialyze each analyzed sample prior to DP voltammetry at the PGE best against phosphate or acetate buffer (see Section Experimental and Ref_ 7 and IZ)_ The results of the study of electrooxidation of DNAs differing in G+C content (Table I) also yield information on the electrode reaction of adenine and guanine residues in DNA at the PGE_ The fact that the ratio of the heights of peaks A and G was always identical with the ratio of the base contents (A+T) I (G+C) for all DNA samples studied can be explained by the fact that the consumption of electrons in the electrooxidation of both adenine and guanine residues in DNA at the PGE is the same under DP voltammetric conditions. EZectrooxidatiops of #roteim

at graphite electrodes

Several proteins yielded a peak even in conventional LS voltammograms in the vicinity of 0-7 V in BRITTOX-ROBINSON buffer, pH 7-4 (Fig. 2). In order to demonstrate the faradaic nature of this protein peak, we electrolyzed a solution of ribonuclease at the WISGE at 0.7 V. After 21 hours of electrolysis, the voltammetric peak of ribonuclease decreased by about 15 Oh_ This result thus indicates that direct electrooxidation of a protein at the WISGE participates in the appearance of

Bnbec

Linear swx-p voitammqgrams of proteins at the WISGE pH ~_a_ (a) I 1-9 @I nhonucle=e, (b) 2.23 @Z albumin.

concentration

of go s[crns.

Voltage

in BRITTOS-ROBISSOS

buffer. (c) histone fraction HI at the scan rate 16.66 mV s-l. initial potential 0.0 V. WISGE

We also recorded cvclic the voltammetric peak of proteins (Fig- 2). voltammo,orams for ribonuclease, albumin, and histone fraction HI 6t a voltage scan rates of r-25 V s-l and pH 7_4_ X11 proteins yielded a peak on the anodic part of the cyclic voltammograms in the vicinity of o.S V. This peak did not have the cathodic counterpart expected for non-faradaic reorientation or adsorptionjdesorption processes [22. 33]_ Moreover, the oxidation peak of the proteins was shifted to more positive potentials with increasing voltage scan rate_ The Iatter two results thus indicate that the electrode process responsible for the formation of the oxidation protein peak (Fig. z) is irreversible_ In order to determine which protein groups are responsible for the electrooxidation current of proteins at the WISGE, we studied first the behaviour of free aminoacids at the WISGE. We found that, of the aminoacids occurring currently in proteins, only tyrosine and tryptophan yielded electrooxidation currents (Fig. 3n.b) in the vicinity of the protein currents at the WISGE. meapeak potential (Fig_ z)_ Electrooxidation surable with difficulty. were yielded also by histidine, methionine, cystine and cysteine. but at potentials approximately 0.4-0.5 V more positive Further details on the behaviour of aminoacids than the protein peaks. at graphite electrodes will be published elsewhere. We investigated further the behaviour of various model peptides at the WISGE_ Poly-L-tyrosine and copolymer poly(L-tyrosine, Ltryphophan) yielded a very well developed voltammetric peak in the vicinity of 0.7 V at pH 7-4 (Fig_ 3c.R). On the other hand, peptides which contained some of the other amino acids o.xidizable at the WISGE except tyrosine and tryptophan (poly-L-histidine, apamin * and glutathion) did not yield any wave or peak in the vicinity of the potential of protein * _.parrin is a peptide of the following aminoacid composition : Cys-Asp (KH,J-Cys-L~s-_~Ia-PO-Glu (NHJ-Thr--AlaLeu-Cys - _AIa-Arg - Arg-Cys-Glu (XH+Gln-His-XH,_ S-S linkages are between Cys I--II and Gys 3-15. The abbreviations for aminoacids were used according with Ref- ~24~

Electrao_xidation

of

i)X_X

and

Proteins

77

yeak (Fig_ 2). The latter peptides yieIded a peak only at potentials close to the oxidation of the background electrolyte, similarly to cystine and histidine, It is therefore probable that the incorporation of an aminoacid o_xidizable at the WLSGE into polypeptide chain does not infiucnce significantly the potential of its oxidation at the WLSGE. These results. together with the results of studies of free aminoacids. enable us to suggest that the o,tidation of tyrosine or tqyptophan residues is responsible for the appearance of the oxidation peak of proteins (Fig 2).

d

0.4

08

r2

a2

0.6

LO

Fig_ 3_ Linear sweep voItammognms of aminoacids and poly(aminoaci&) at the l\X5GE in (a) I x IO-Q Jl tyrosine ; (b) L x IO--~ Jf tryptophan ; BRITTOS -ROBISSOX buffer. pH 74 (G) z-6 x ro-5 31 poly-l-tyrosinc ; (ri) 1.5 2; LO-~ copolymer poly(t-t~~sine, L-tc?tophan) The poly(aminoacids) concentration IS related (tyrosine L tryptophan ratio WLS 4 : I)_ to the monomer content. (n), (6) WISGE Xo. z ; (c). (n) W’ISGE Xo_ r and initial potential 0.0 v_

In order to obtain better defined voltammetric peaks of proteins at the WISGE, we used, instead of LS voltammetry, the more sensitive DP voltammetry. The advantage of using DP voltammetry for the protein analysis at the WISGE follows from a comparison of Figs. z and 4. DP voltammetry was employed in a study of the influence of pH on the oxidizabiiity of proteins at the \VTSGE_ Albumin. ribonuclease and insulin (i-e_ proteins containing no or relatively few tryptophan residues as compared with the content of Qrosine residues) yielded only a single o_xidation DP voltammetric peak at the WISGE (Fig. 4rs-c). Lysozyme, which contains twice as many tryptophan residues than the tyrosine ones, yielded at pH 7.4 besides this peak another one at potentials by cn 50 rnlr We compared the peak potentials (U,) of the more positive (Fig. 4d). two Iysozyme peaks with 0, of monomeric tyrosine and tryptophan in a broad range of pH (z.5-7.0) (Fig. 5). VJe found that the potential

Brabec

t

a2

0.6

L2

I

Differential-pulse X-oltammo,sams of buffer, pH 7-0~ (a) 7-9 @Z ribonuclease. Iysozyme_ WISGE Xo_ I_

proteins (b) x-12

at +%I

the WISGE in BRITTOS-ROBISSOX albumin : (c) 4-1 y-31 insulin : (n) 7-z y.M

of the more negative peak of lysozyme was indent&l with U, of tyrosine, and the potential of the more positive peak of lysozyme was identical with U, of tryptophan- The peak potentials of albumin and ribonuclease were always identical with U, of tyrosine, Other proteins investigated yieIded only one peak at U, of tyrosine (Fig. 4a-c) evidently because they either did not contain tryptophan residues at all (ribonuclease, insulin), or because they contained only very few tryptophan residues as compared with the tyrosine ones (albumin), so that the relatively small oxidation current of tryptophan residues disappeared in the substantially higher current of tyrosine residues_ It can therefore be summarized that even the results of DP voltammetry of proteins at the WISGE agree with the conclusion according to which oxidation of tyrosine or tryptophan residues is responsible for the appearance of the oxidation peaks of proteins (Figs. 2 and 4)_ The dependences of the protein peak heights on the protein concentration tend to a limiting value (Fig. 6) suggesting a participation of the protein adsorption in the electrode process. The latter conclusion is also supported by a linear character of the dependence of the LS voltammetric peak current of proteins on the voltage scan rate. The lower level of the analytical utility of DP voltarnmetry of proteins at the WISGE is ca o-5 &cm=_

Electrooxidation

of DXA

and Proteins

79

0.8 -

.

I

3

PH

1

s

7

Variation of the differential puke-x-altammetric peak potential UP_ with pH for tyrosine and trpptophan and for peaks of lysocyme. The concentration of tyrosine and tryptophan was r Xx0-* :\I. lysoryme concentration was 7-a cl_lf_ Curve -4 represents the linear UP us. pH relationship for tyrosine (esperimental points not shown). while cm-x-e B represents the linear UP LX pH relationship for tryptophan (experimental points not show-n)_ The esperimental points for more negative peak of lysoryme are represented by solid circks (a) ; for more positive peak of Iysozyme the experimental points are represented by open WlSGE No_ I_ cirde5 (0).

D-

o-

CoGf)

;+G-=+

0 -.

Fig. 6. Variation of the relative differential pulse-voItammetric peak height y with the concentration of the protein at the WISGE in

O-

BRITTOS-ROBISSOS buffer. pH 7_+ (a) ribonuclease; (b) albumin.

I

0

I

1

I

2

I

3

COJM) I

4

The peak height of 23-5 pdf ribonuclease and 4-5 PM albumin was taken as roe X_ WISGE x0. I_

We endeavoured to tznd out whether the denaturation of the protein with acid or urea would be manifested by a change in the DP-voltammetric behaviour of the protein at the WISGE. We found that the DPvoltammetric peak of ribonuclease in 0.1 N H&O, was by ca 30 o/0 higher than that obtained at pH 7.4. The addition of 8 .ii urea to a neutral solution of ribonuclease led to an increase of the ribonuclease peak even by about 40 y. (Fig. 7). These results indicate that ribonuclease changes

Fig. 7. Variation of the relative differential @se-voltammetric peak height of 6.6 @I rihonuclcase 2 with urea concentration in Ekxurros-ROBISSOX buffer. pH 7-4. The peak height of 6.6 ydl ribonuclease without urea was taken as mo X_ WISGE Xo. I.

its conformation after the addition of S 111urea or in 0.1: N H2SOI in such a way that the number of its tyrosine residues accessible for the reaction with the graphite electrode is significantly increased_ This conclusion agrees with the results of non-electrochemical studies aimed at the detection of tyrosyl residues buried inside the ribonuclease moIecule or It can be therefore expected that DP exposed to the environment [q]. vokrmmetry of proteins at graphite electrodes will also become a method suitable for studies of conformational changes of proteins.

The first results of the electrochemical analysis of biopolymers were obtained by means of classical polarography. However, this method was too insensitive for investigations of oxidation-reduction properties of biopolymers. The low sensitivity of this method for the analysis of polyrwric substances was caused by the reIatively low diffusion coefficients of macromolecuks as wel1 as, in some cases, by the low accessibility

Electrooxidation

of DNA

81

and Proteins

of the majority of the electroactive groups of a pol_ymer for the interaction with electrode, because they were buried inside the molecule_ An increase in sensitivity was achieved by using modem, more sensitive methods of electrochemical analysis (oscillopolarography. pulse polarography) as we11 as by exploiting stationary H.M.D.E.. enabling the accumulation of the polymer at the e1ectrode surface over a prolonged time by polymer adsorption_ The use of mercury electrodes limits the experimenter namelly to studies of electroreduction and adsorption of biopolymers and, in the case of proteins, also to the exploitation of BRDI~KA’S reaction. The results of the present study show that the use of graphite electrodes in connection with DP voltammetry considerably widens the field of applicability of electrochemistry in the investigation of biologically important macromolecules. From this point of view, one can consider, as especially important results. not only the possibility of studying A-T and G-C pairs in nucleic acids independently, but also the possibility of investigating by means of electrochemical methods proteins that do not contain cyst&e, cysteine, or an electroactive prosthetic group_ I-&tones, which may serve as genetic repressors of DNA in chromatin, belong to important proteins that have not yet been made analyzab1e by methods using mercury electrodes_ The voltammetric study of free b&tones as well as of histones bound in chromatin at graphite electrodes could bring new information which would throw hght on their important biological function-

Acknowkdgcment The

skilful

BIr VOJTECH

technical

UORXSTEIN

assistance of Mrs IREXA POSTBIEGLOV,~ and is gratefullyappreciated_

References [I] H_\V. XGRSBERG Systems.

and P_ VALEXTA. in Ions isz Mucronrobcrrlar aard BioZo&aZ and B. VISCEXT (Editors), Scientechnica, Bristol

D-H_ EVERETT

(xg$3), p. 201. 123 V_ BRABEC and E. PALEC~EK, J_ Eiecfroanal [3]

88 (1978) 373 V. BRABEC and

[4]

J-A_ them.

[5] E.

REYNAUD. Bioener,s

Chenr.

Infeerf&aZ

E. PALEC~EK, Biopolymers 11 (~972) 2577 B. BLXLFOY. J-M_ SEQU_ARIS and P-J_ SKCARD. 4

(1977)

380 in lVzrcZeic Acid

Electrochem.

Bioelectro-

PALE~EK. in Progmss Researclt aud Molecular W-E_ COHN (Editor). Academic Press, Xew York (1976). Vol. 18. [6] H_ BERG. in Topics in Bioelecivochemisfvy awed Bioenev,eeticss, G(Editor). Wiley-Interscience. Xeew York, London (1976). Vol. I,

BioZo,oy.

p_ 151 MIIAZZO

p_ 39

S-2

Brabec

ET]

V_

(S]

89 (1975) E. PALE~EK

[IO] (II] (121 [13] (14] [IS] [16) ir+J HIS] Erg] fzo>

[-I] 1221 [23] E2+] (23:

J_ Electrond.

Ckeriz. ZnterfatiaZ

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