Biochimica et Biophysica Acta, 412 (1975) 157-167 © Elsevier Scientific Publishing Company, Amsterdam - - P r i n t e d in The Netherlands BBA 37184

STUDIES ON ELECTRON TRANSFER BETWEEN MERCURY ELECTRODE AND HEMOPROTEIN

F. SCHELLER a, M. J ~ N C H E N a, J. LAMPE a, H.-J. PR1]MKE a, J. BLANCK a and E. PALECEK b

aCentral Institute of Molecular Biology of the Academy of Sciences of the G.D.R., Department of Biocatalysis, Berlin-Buch, Lindenberger IVeg 70, 1115 (Germany) and blnstitute of Biophysics of the Czechoslovak Academy of Sciences, Brno 61265 (Czechoslovakia) (Received April 29th, 1975)

SUMMARY

The electrochemical behaviour of ferricytochrome c, metmyoglobin and methemoglobin was studied using d.c., a.c. and differential pulse polarography, and controlled potential electrolysis. 1. The three hemoproteins yield d.c. polarographic steps, and peaks in differential pulse polarograms, the height of which is proportional to concentration. The charge transfer is influenced by strong adsorption. 2. The concentration dependence of the a.c. polarograms indicates structural changes in the adsorbed molecules. 3. The reduction products of controlled potential electrolysis of metmyoglobin and methemoglobin have absorption spectra identical with the native control samples. The affinity for oxygen and the cooperativity in hemoglobin are not affected by the reaction at the electrode. 4. The charge transfer proceeds via adsorbed, already reduced, molecules to freely diffusible proteins.

INTRODUCTION

Methods of electrochemical analysis have shown their usefulness in biochemical research, e.g. for quantitative determination of biologically active substances [1-3], for characterization of reducible groups and structure of biopolymers [4-7], for investigation of conformational changes [8-13], and for amperometric titrations of mercapto groups [14]. Polarography has been recently successfully applied in nucleic acid structure analysis [4, 5]. For the polarographic analysis of proteins mostly the Brdicka's medium containing cobalt salts was used [1, 3, 8]. In these media proteins produce catalytic currents; the mechanism of the electrode processes responsible for these currents is not yet fully understood. The non-catalytic polarographic reduction currents of proteins (a reduction of-S-S- groups [15-17] and other reduction sites present, e.g. in prosthetic groups [18-23] have been studied to

158 a much lesser extent in spite of the fact that this type of studies has many advantages to the complex catalytic processes. The former studies may yield a number of important electrochemical parameters which can be utilized in the elucidation of both the electrode process and the mechanism of biological redox reactions. Recently attention has been paid to the hemoprotein interactions with the electrode. It has been shown that the free prosthetic group of hemoproteins, Feprotoporphyrin IX is reversibly reducible on the mercury electrode in aqueous solution [24, 25] and mixed ferrous ferric intermediates occur at cathodic reduction [26]. Hemin in alkaline solution and the hemin-dicyano complex [27] yielded heterogeneous rate constants ks showing the extreme rate of charge transfer. The influence of the protein moiety upon the electrochemical behaviour of heroin was simulated in several studies by complexing agents, or certain solvents [28-30]. It was shown, that z-axis ligands have a significant effect on the thermodynamics of electron transfer and that modification of the Fe spin state by the ligand can moderate the rate of electrode reaction as well [27]. Studies on the redox behaviour of proteins were usually made by means of potentiometric and indirect coulometric titrations [31] and pulse radiolysis [32]. Brown [33] and Gygax and Jordan [34] reported that the prosthetic group in the trivalent state in the hemoproteins, the hemin, fails to yield a polarographic reduction step. On the contrary Betso and coworkers [19, 20] have recently shown a welldeveloped step for methemoglobin and for horse-heart cytochrome c. In this paper we have confirmed the finding of Betso and coworkers [19, 20] and shown that beside methemoglobin and cytochrome c, metmyoglobin too produces a d.c. polarographic step. We have studied all these three hemoproteins by various electrochemical techniques, and obtained data concerning the protein interaction with the electrode including electron transfer and nativity of reduction products. MATERIALS AND METHODS The background solution was prepared from twice recrystallized KC1 or phosphate buffer in bidistilled water. The pH was adjusted by addition of HC1 or KOH, respectively. Dissolved oxygen was removed by bubbling the solution with purified N2. The horse-heart cytochrome c investigated was purchased from Reanal, Budapest. Bovine metmyoglobin and human methemoglobin were prepared by standard methods. For a.c., d.c. and pulse polarography, capillaries with about 3 s drop-time were used. A mercury-pool electrode of about 4 cm 2 surface served as working electrode in preparative electrolysis. All potentials were related to the saturated calomel electrode. The temperature of the vessel was maintained at 25 ~: 0.1 °C. The solution volume varied from 2 to 20 ml. The d.c. polarographic measurements were performed with an OH 102 polarograph (Radelkis, Budapest). For pulse polarographic studies a polarographic analyser PAR 174 (Princeton Applied Research, Princeton, U.S.A.) was used. The a.c. polarograms were measured with a GWP 563 polarograph (AkademieWerkst/~tten, Berlin-Adlershof, G.D.R.). The a.c. frequency was 78 cps ; the amplitude was adjusted to 10 mV. In the absence of faradaic processes the recorder reading was proportional to the capacitance of the electrode. The dependence of relative decrease

159 of capacitance A C/Co on drop time tm and concentration c, respectively, was evaluated according to Eqn 1, which holds for diffusion limited adsorption processes [35]:

(1)

d C/C0 = 0.77.7.36" l O- 4. S" D 1/~"NL" c" tm a/z" A Cs/(~0

D, diffusion coefficient ( c m Z ' s - 1 ) ; S, surface area (cmz per molecule); ACs, capacity decrease at full coverage of the electrode; NL, Loschmidt's number. The preparative electrolysis was carried out with a PS 2.3 potentiostat (Forschungsinstitut Meinsberg, G.D.R.). After cathodic reduction of methemoglobin, deoxyhemoglobin was transferred into tonometric cuvettes under aerobic conditions. To remove oxygen oxyhemoglobin was deoxygenated with nitrogen. Titration was performed by stepwise addition of air. The degree of oxygenation was determined spectrophotometrically. The oxygen partial pressure at 50~ saturation and the sigmoid coefficient were calculated on a computer R 300 (VEB Kombinat Robotron Dresden) by means of an ALGOL program. The absorption spectra of solutions were measured by means of a Unicam SP 700 spectrophotometer. The kinetics of azide binding to partially reduced methemoglobin was performed by means of a DURRUM stopped flow device. RESULTS AND DISCUSSION

d.c. and pulse polarography

Cytochrome c, methemoglobin and metmyoglobin produce well-defined reduction steps (Fig. 1). The half-step potentials E1/2 for these hemoproteins are considerably more negative than the redox standard potentials, and are shifted in the cathodic direction with rising concentrations. The limiting current lu,, in equinormal solutions with respect to hemin (val/1) differs substantially for the three proteins.

~[.A] (~3

if2

0.1 y o

0.5

1.0

1.5

Iv]

Fig. 1. d.c. polarograms of hemoproteins. 25 °C, 0.l M phosphate buffer, concentration 0.l mval/l. 1, ferricytochrome c, pH 6; 2, methemoglobin, pH 6; 3, deuterohemin, pH 11.5; 4, metmyoglobin, pH 6.0.

160

o.os

o

0:5

i

c.10~ [ v ~ ]

Pig. 2. Concentration dependence of the polarograpbi¢ limiting cu~ye~ts, l, deuterobemi~; 2, metbemoglobin; 3, metmyoglobin; 4, ferficytochrome c. Same conditions as in Pig. 1.

For cytochrome c the limiting current and the half-wave potential are strongly dependent on pH [20]. In the neutral region the limiting current has the maximum value and decreases to about one half at pH 8 and pH 3. Below pH 6.5 metmyoglobin yields a polarographic step the height of which rises to the double value from pH 6.5 to 5.0 but the half-wave potential is nearly constant. The d.c. step of methemoglobin has nearly constant height and half-wave potential between pH 5.5 and 8.0. The height of the d.c. steps depends linearly on concentration of the three hemoproteins in the region from 0.05 to 0.3 reval/1 (Fig. 2). At pH 5 the limiting currents of metmyoglobin reduction correspond to the diffusion coefficient. In contrast the maximum D value of cytochrome c from Ilkovic equation is approximately the half of D value determined by hydrodynamic methods. A comparison with the limiting currents for hemin shows that the methemoglobin reduction process approximately obeys the Ilkovic equation. From this it results that four electrons are taken up per molecule of methemoglobin. At high concentrations, the reduction step of methemoglobin is distorted into a peak, and the concentration dependence of the limiting current deviates from linearity. The sensitivity of the d.c. polarographic method is limited by the magnitude of the capacity current. In pulse polarography the capacity current is virtually eliminated and the sensitivity of this method is considerably higher as compared with d.c. polarography [36]. The former method is therefore highly suitable for the analysis of high molecular weight substances (with low diffusion coefficients) which

161 d

t O,A]

Ca) S

t[~]

0.2

O~OS

o.i 1

o~

,,-e

u'~

oe

D

0

_°]s[~l

~s~s~?~ ®-sfv] L

o~

Fig. 3. Differential pulse polarograms of methemoglobin. 0.1 M phosphate buffer, pH 7, 25 °C. (a) Concentration dependence, d = 50 mV, scan rate 2 mV/s, drop time 2 s. 1, background solution; 2, 0.01 mval/l methemoglobin; 3, 0.04reval/1 methemoglobin; 4, 0.07mval/1 methemoglobin; 5, 0.10 reval/1 methemoglobin. (b) Dependence on pulse amplitude d. 0.07 mval/1 methemoglobin. 1, 5 mV; 2, 10 mV; 3, 25 mV; 4, 50 mV; 5, 100 mV.

Jp

[pA]

0.10

~

o.o,

o

0.25

sl°

.

0.s

0.35

1?od[pv] 1.oo ~.~0 ~ [v~/~

~iB. 4. Dependence of ~eak cu~ent ~, on concentration and pulse amplitude d, ~spectiv~ly, for metbemoBlobin. Same condlt~ons as in ~i~. 3. I, eon~nt~afion dependence, d = 50 mV; 2, dependence on ~ul~e amplitude, 0.07 m~al/I metb~mo~ob~n.

162

[/aF]

0.3

0.2

i

I

0.5

1.0

t

1.5

~

-E[v]

Fig. 5. a.c. polarograms of metmyoglobin. 0.1 M KC1, 25 °C, tm= 3 s. Metmyoglobin concentrations in/zM: 1, 0; 2, 0.6; 3, 1.2; 4, 1.5; 5, 1.9; 6, 2.2; 7, 2.7; 8, 3.2; 9, 6.0. produce only very small d.c. polarographic currents. Methemoglobin and cytochrome ¢ produce well-defined peaks whose summit potentials coincide roughly with E~/z of the d.c. polarographic steps (Fig. 3). The peak height increases linearly with the protein concentration (Figs 3a and 4) and the pulse amplitude (Figs 3b and 4). The increase in concentration results in the shift of the summit potential to more negative values as well as in the increase of the half-width of the peak. a.c. polarography

As generally observed with proteins [37], adsorption of metmyoglobin causes a sharp decrease of the electrode differential capacitance in the vicinity of the zero charge potential (Fig. 5). Although the d.c. polarographic steps are found in the adsorption area, corresponding peaks for the reduction of the prosthetic groups do not occur in the a.c. polarograms. In accordance with the shift of El~ 2 with the protein concentration, and the slope of the d.c. polarographic steps this behaviour shows the irreversibility of electrode process. Evaluation of the concentration and drop-time dependence of the a.c. polarograms in the adsorption region shows that the relative decrease of capacitance A ~'/~'0 depends linearly both on concentration and tm ~/2 (Fig. 6). Under these conditions the surface S occupied by one protein molecule at the electrode can be calculated by Eqn 1. In contrast to the pH dependence of limiting current of metmyoglobin the S value does not alter in the region from pH 7 to pH 4.5 (Fig. 6). The S values are considerably larger for the three proteins than the largest cross-section of the native molecule in the crystal (Table I). From this finding, Birdi's [38] results obtained at the water-air interface, and Kuznetsov's [13] studies in Brdicka's solution we concluded that the globular proteins adsorbed at the electrode are unfolded [37]. The value of 20/k 2 per amino acid residue obtained for the three hemoproteins under

163

a 0.5 /%/-

-- __

'II 'X

I1' ~:

0./+

•

2///

z/z /

0.3 0.2 0.1

/ I

I

I

I

I

I

I

1

2

:3

/.

5

~

~

c,

°

~

[Mot/t]

Fig. 6. Evaluation o f a.c. p o l a r o g r a m s o f m e t m y o g l o b i n . 0.1 M KC1, 25 °C, tm = 3 S, E = - - 7 0 0 mV. 1, p H 7.0; 2, p H 4.5.

investigation is in very good agreement with that calculated from X-ray diffraction for unfolded proteins [39]. These suggestions of structural changes due to adsorption at the electrode raised doubts whether the proteins in solution reduced in a cathodic reaction remain in the native state.

Macro-scale electrolysis The cathodic reduction of methemoglobin at the plateau of the d.c. step (--1 V vs S.C.E.) leads to the formation of the oxyhemoglobin spectrum (Fig. 7). This is parallelled by the decrease of the d.c. polarographic reduction step. At a current of 60 ,uA within 5 h, 2 #mol of protein were completely reduced to hemoglobin, which reacts to give oxyhemoglobin on exposure to air. In the visible spectral region polarographically reduced deoxyhemoglobin and, after exposure to air, oxyhemoglobin do not differ from native control samples. Location and shape of the absorption maxima are identical (Fig. 7). The oxygen half pressure at p H 6.65 was determined to be 14.5 Torr. The sigmoid coefficient was calculated to 2.5. For the control samples 15.2 Torr and 2.6, respectively, were obtained (Fig. 8). TABLE I V A L U E S O F R E D O X P O T E N T I A L S Eo', H A L F - S T E P P O T E N T I A L S E~ (0.1 m M ) , D I F F U S I O N C O E F F I C I E N T S D, A N D S U R F A C E S F O R H E M O P R O T E I N S

Cytochrome c Metmyoglobin Methemoglobin

SHg/w~tcr Serystal

Eo' E~ ( m Y vs S.C.E.)

D D.kovi~ (cm2.s -1 × 10 6)

(A2/subunit)

20 --190 -- 90

0.95 1.26 0.95

2100 3500 2850

-- 130 --1050 -- 600

0.20 0.70 1.00

1200 1600

164 Extinction

7 ~

,1 2 3

I

~ 6

I

2O

18

!

~.

10_3 [cm_t]

----

16

Fig. 7. Visible absorption spectra of 0.2 mval/1 methemoglobin solutions at successive stages of controlled potential reduction at --1000 mV vs saturated calomel electrode.

The electrochemical preparation of partially reduced methemoglobin reported by Betso and Cover [19], whose species contained Fe(II) and Fe(III) in a molecule could not be reproduced. As shown in Fig. 2 the overall number of electrons taken up per molecule is four, that is, all four hemins of one methemoglobin molecule are reduced. To confirm this result the kinetics of azide binding to the oxidized molecules within the partially reduced methemoglobin solution was investigated. Because of the chain specificity of the ligand binding process on native methemoglobin [40, 41] any chain specific reduction should be detected. The behaviour of the partially reduced

,oo

/

°

/ 1/,,2

Y

-1

li f

0.5

I

~

~.0 1,5 log p02

I

~,0

Fig. ~. Hill p]ots of o×y~e~ s ~ f i o o ~/).

cuffed. 0.05 ~ ~bo~b~te b~e~, ~ 6.65, c ~ o ~ ,

1, elect~ocbe~]c~Hx ~ed~ced ~etbe~o~ob~; 2, be~o~lobJ~ coat,o].

= 0.3)5

165 methemoglobin corresponded to that of the controls methemoglobin and mixtures of methemoglobin and oxyhemoglobin in showing two chain specific reactions of equal amplitude. The difference of about 40 mV between the redox potentials of the a and the fl chains [42] is evidently too small for a chain-specific reduction in comparison with the high cathodic overvoltage. The final product of the electrolysis at --1.3 V is identical with that reduced at --1 V. Analogous results were obtained for metmyoglobin; potential-controlled electrolysis at --1.2 V yields the reduction product oxymyoglobin. By autoxidation of oxymyoglobin small amounts of metmyoglobin are regenerated, causing a little change in the spectrum. Oxidation of the electrolysis product with ferricyanide leads to native metmyoglobin.

Mechanism of electron transfer Comparison of the results obtained by the different methods suggests that the electrode reaction with hemoproteins is the electron transfer from the electrode to the prosthetic group: In d.c. and pulse polarography the reduction current is proportional to the concentration of heroin in proteins and the preparative electrolysis supplies proteins which contain heme. The concentration dependence of d.c. and a.c. polarograms shows that the limiting current increases linearly with concentration also if the electrode has been fully covered with protein molecules. Consequently, a much higher charge is transferred at one mercury drop than corresponds to the reduction of one monolayer. This concentration dependence of the faradaic current presupposes either a very rapid exchange of adsorbed molecules or the charge transfer through several adsorption layers. Studies on the adsorption behaviour of proteins have shown that proteins near the zero charge potential are irreversibly adsorbed at the mercury electrode [13]. The concentration dependence of the reduction of disulfide groups in the proteins ribonuclease, bovine serum albumin, trypsin, and insulin does not indicate either any rapid exchange of adsorbed molecules with freely diffusing particles. At saturation of the electrode, the limiting current reaches a maximum [16, 17]. It seems therefore more probable that the electrons are transferred through already reduced adsorbed molecules to the proteins reaching the adsorption layer. It is likely that the electrons on rotation of the protein molecules are transferred between the proteins molecules via the exposed heme edge. However, the charge transfer might go also through areas of the protein fabric. In both cases the overall rate of the reduction process is determined by the transport of oxidized proteins to the electrode, and by the charge transport across the adsorption layer. The latter of these two processes may lead to the pH dependence of the limiting current, to the too low D values according to the Ilkovic equation, and to variation of the half-step potential with the protein concentration. The linear dependence of the height of the pulse polarographic peak on the pulse amplitude (Fig. 4) is in agreement with that observed with simple inorganic depolarizers [43] as well as with peaks yielded by proteins in Brdicka's cobalt solution [2] and differs from that of nucleic acids [4446]. This difference is not surprising if we take into consideration the potential range in which methemoglobin is adsorbed at the electrode (Fig. 5) and its reduction potential (Figs 1 and 3); the desorption

166 potential o f the p r o t e i n differs t o o much f r o m the reduction potential to cause the n o n - l i n e a r i t y o f the d e p e n d e n c e observed with nucleic acids. A n essential c o n d i t i o n for biochemical utilization o f the cathodic reduction o f p r o t e i n s is the generation of native reduction products. Betso et al. [20], perf o r m i n g a cathodic reduction o f f e r r i c y t o c h r o m e c, o b t a i n e d a p r o d u c t the s p e c t r u m o f which indicates changes o f the t e r t i a r y structure; however, it exhibited a p p r o x i m a t e l y the same enzymatic activity as the m a t e r i a l reduced with dithionite. The optical spectra o f the p r o d u c t s of c o n t r o l l e d p o t e n t i a l electrolysis o f m e t h e m o g l o b i n a n d o f m e t m y o g l o b i n were identical with the c o r r e s p o n d i n g native proteins. By means o f the oxygen binding we were able to d e m o n s t r a t e t h a t the reduction o f m e t h e m o globin at the m e r c u r y electrode yields native t e t r a m e r i c hemoglobin. The u n c h a n g e d oxygen affinity a n d the Hill coefficient c o m p a r e d with the native h e m o g l o b i n suggest no structural changes. The nativity o f the reduction p r o d u c t s is n o t in c o n t r a d i c t i o n to the discussed structural changes u p o n a d s o r p t i o n , which were j u d g e d f r o m the a.c. p o l a r o g r a m s . In the p r o p o s e d m e c h a n i s m o f electron transfer, only a negligibly small p o r t i o n o f the c o n v e r t e d proteins is in direct contact with the p o o l electrode. The m a j o r i t y o f molecules is c o n v e r t e d at a p r o t e i n - s o l u t i o n interface. This interface should n o t cause any drastic irreversible structural changes in g l o b u l a r proteins.

REFERENCES 1 Brezina, M. and Zuman, P. (1956) Die Polarographie in der Medizin, Biochemie und Pharmazie, Akademie Verlagsges. Geest und Portig, Leipzig 2 Palecek, E. and Pechan, Z. (1971) Anal. Biochem. 42, 59-71 3 Homolka, J. (1971) in Methods of Biochemical Analysis (Glich, D., ed.), Vol. 19, pp. 435-555, John Wiley and Sons, New York 4 Palecek, E. (1971) in Methods in Enzymology (Grossmann, L. and Moldave, K., eds), Vol. 21, pp. 3-24, Academic Press, New York 5 Palecek, E. (1969) in Nucleic Acid Research (Daridson, J. and Cohn, W., eds), Vol. 9, pp. 31-73, Academic Press, New York 6 Scheller, F. and Will, H. (1973) FEBS Lett. 29, 47-50 7 Berg, H., Granath, K. and Nygard, B. (1972) J. Electroanal. Chem. 36, 167-178 8 Mfiller, O. (1963) in Methods of Biochemical Analysis (Glich, D., ed.), Vol. 11, pp. 329-403, John Wiley and Sons, New York 9 Berg, H. (1968) Chim. Phys. 65, 54-58 10 Scheller, F. and Jehring, H. (1972) Studia Biophys. 33, 211-221 11 Ruttkay-Nedecky, G. and Bezuch, B. (1971) Experientia Suppl. 18, 553-562 12 Kalous, V. (1971) Experientia Suppl. 18, 349-354 13 Kuznetsov, B. (1971) Experientia Suppl. 18, 381-386 14 Cecil, R. and Wake, R. (1962) Biochem. J. 82, 401-406 15 Theorell, H. (1938) Biochem. Z. 298, 258-260 16 Cecil, R. and Weitzman, P. (1964) Biochem. J. 93, 1-11 17 Pavlovic, O. and Miller, J. (1971) Experientia Suppl. 18, 513-524 18 Weitzman, P., Kennedy, J. and Caldwell, R. (1971) FEBS Lett. 17, 241-244 19 Betso, S. and Cover, R. (1972) J. Chem. Soc. Chem. Commun. 1972, 621 20 Betso, S., Klapper, M. and Anderson, L. (1972) J. Am. Chem. Soc. 94, 8197-8210 21 Scheller, F., J~ihnchen, M., Etzold, G. and Will, H. (1974) Bioelectrochemistry 1,478-486 22 Ramasamy, N., Ranganathan, M., Duic, L., Srinivasan~ S. and Sawyer, P. (1973) J. Electrochem. Soc. 120, 354-361 23 Redwood, W. and Godschalk, W. (1972) Biochim. Biophys. Acta 274, 515-527 24 Bednarski, T. and Jordan, J. (1967) J. Am. Chem. Soc. 89, 1552-1558

167 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Montalvo, Jr. J. and Davis, D. (1969) J. Electroanal. Chem. 23, 164-166 Kadish, K. and Jordan, J. (1970) Anal. Lett. 3, 113-122 Kadish, K. and Davis, D. (1973) Ann. N.Y. Acad. Sci. 206, 495-503 Davis, D. and Martin, R. (1966) J. Am. Chem. Soc. 88, 1365-1371 Cauquis, G. and Marbach, G. (1974) Bioelectrochemistry 1, 23-28 Kassner, R. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2263-2267 Steckhan, E. and Kuwana, T. (1974) Ber. Bunsenges. 78, 253-259 Pecht, J. and Faraggi, M. (1972) Proc. Natl. Acad. Sci. U.S. 69, 902-906 Brown, G. (1954) Arch. Biochem. Biophys. 49, 303-318 Gygax, H. and Jordan. J. (1968) Discuss. Faraday Soc. 45, 227-233 Jehring, H. and Horn~ E. (1968) Monatsber. Dtsch. Akad. Wiss. Berlin 10, 295-302 Flato, J. (1972) Anal. Chem. 44, 75A-87A Scheller, F., J~ihnchen, M. and PriJmke, H.-J. (1975) Biopolymers, in the press Birdi, K. (1972) Kolloid Z. Z. Polym. 250, 222-226 Yamashita, T. and Bull, H. (1967) J. Colloid Interface Sci. 24, 310-315 Blanck, J. and Scheler, W. (1968) FEBS Lett. 4, 217-221 Gibson, Q., Parkhurst, L. and Geraci, G. (1968) J. Biol. Chem. 244, 4668-4676 Banerjee, R. and Cassoly, R. (1969) J. Mol. Biol. 42, 337-349 Parry, E. and Osteryoung, R. (1965) Anal. Chem. 37, 1634-1637 Palecek, E. (1972) Collect. Czech. Chem. Commun. 37, 3198-3208 Brabec, V. and Palecek, E. (•973) Z. Naturforsch. 28c, 685-692 Palecek, E. and Doskocil, J. (1974) Anal. Biochem. 60, 518-529