Proton conductivity in the solid hydrated haemoglobin

BIOCHIMICA ET BIOPHYSICA ACTA

293

BBA 4 5 0 1 4

PROTON CONDUCTIVITY IN THE SOLID HYDRATED

HAEMOGLOBIN

S. M A R I ( ; I C , G R E T A P I F A T AND V. P R A V D I ( ;

"Ruder Bo,¢kovid" Instilule, Zagreb (Jugoslavia) (Received J u l y 2nd, t963)

SUMMARY

I. Solid state electrolysis experiments were used to study the interaction of water vapour with bovine methaemoglobin. 2. With 9.17 % water adsorbed, which is equivalent to about one and a half times the monolayer amount, there is no proton conduction, presumably because of lack of continuous hydrogen-bond chains. 3- The existence of a hydration shell with such chains is indicated by the preponderance of proton conduction at a coverage corresponding to three monolayers. 4. The measured energy of activation, 0. 7 eV, is of the order encountered in protonic semiconductors. 5. The dependence of the dielectric constant on hydration is also in agreement with this model. 6. Permanent polarization after electrolysis of the 18 % hydrated haemoglobin is ascribed to proton deficiency in the hydrogen bond network after electrolysis. 7. At higher coverages, when "liquid" water is present between hydrated haemoglobin molecules, polarization decays in a few minutes. It is postulated that in this case a restoration of the initial state is accomplished by diffusion of protons from the "liquid" water into the hydration shell.

INTRODUCTION

A considerable body of experimental data on electrical conductivity in biologically important substances has been accumulated. It emerges that semiconductor properties, i.e. an increase in electrical conductivity with temperature, is quite a common phenomenon. PULLMAN AND PULLMAN1 pointed out recently that it is not yet clear whether the semiconductivity in biological substances is electronic, ionic, caused b y impurities, or whether it is a combined phenomenon. The question becomes important when approaching conditions in vivo and is relevant in particular to the interaction of biological macromoleeules with molecules of water. However, most of the available data refer to the dry or nearly dry state of materials used for electrical conductivity measurements, although there are instances in which the influence of adsorbed water was studied. An up to date and comprehensive review of the field is due to EI.EY 2, who also presents a considered opinion regarding the role of hydration, suggesting that at higher coverages of adsorbed water proton conductivity m a y take over. In the same book DOCHES~E3 also directs Biochim. Biophys. Acta, 79 (1964) 293-3 ° °

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s. MARI~I~, G. PIFAT, V. PRAVDI~

attention to the importance of adsorbed water in connection with other interesting properties of biological macromolecules such as piezo- and ferro-electricity. ELEY AND SPIVEY4 reported measurements of electrical conductivity in hydrated haemoglobin. They suggest that proton conduction sets in after the physical surface has been filled with adsorbed water, but no direct proof has been produced. ROSENBERG5 on the other hand claims such a direct experiment with a negative result. The same author made further systematic measurements 6 which led him to the conclusion that the sole influence of the water adsorbed on haemoglobin was to increase the dielectric constant of the protein and thus to lower the work necessary to separate the charges due to the increase in the polarizability of the medium. However, we felt that no final conclusion could be drawn before a solid state electrolysis experiment had been made on haemoglobin with different degrees of hydration.

EXPERIMENTAL

Material We used bovine haemoglobin which according to the producer (Biochemical Research Corporation, California), is prepared after DRABKIN~, and is "IOO % pure by electrophoresis". No further treatment was undertaken before the measurements. The methaemoglobin content was checked spectrophotometrically and was found to be 88 % in the original haemoglobin and 97-1oo % in the hydrated samples. The original sample lost 3.73 % of its weight, presumably adsorbed water, on evacuation (Io -4 m m Hg) over P~O5 at 75 °. Several samples of the original haemoglobin were exposed in desiccators at 22 + I ° to different relative humidities over sulphuric acid solutions. Some of them were left for a few months, and occasional weighing indicated the attainment of equilibrium. From the original and hydrated samples, sandwich pellets (graphite/haemoglobin/graphite) of 13 m m diameter were prepared by pressing at IOO kg/cm 2. The weight of haemoglobin was 0.20-0.22 g and the thickness of the discs varied between 1.25 and 1.3o ram.

The electrolysis cell The pellets were cemented (using "Araldite" polyester cement) as quickly as possible between two halves of a cell described earlier 8, but improved in two respects (see Fig. i). Instead of following the movement of the mercury meniscus in the capillary by a cathetometer (i.e. visually), we measured the corresponding change in resistance of a tungsten wire (diameter 50/~) stretched within the capillary. Plots of resistance vs. volume of capillary were obtained by weighing the mercury dropped out at different points of the capillary. Also, in the present cell the whole measuring length of the capillary was thermostated. A high-speed circulating ultrathermostat was used and the temperature was kept constant to within o.I °. The following procedure was used in checking whether the gas evolved during the electrolysis was hydrogen. A tape of filter paper was immersed in an acidified PdCI 2 solution. The other end of the tape was placed over the open end of the capillary. A black spot due to reduced palladium formed if the evolved gas was hydrogen. A blank tape exposed to air was simultaneously kept for control. Biochim. Biophys. Acta, 79 (1964) 293-3 oo

PI{OTON CONDUCTIVITY IN SOLID HYDRATED HAEMOGLOBIN

205

In such an experiment there is only a small amount of mercury left, thus securing electrical contact but leaving free escape of gas through the capillary.

I \

"

"'Tungsten

wire

0

5 cm

Fig. I. The glass electrolysis cell (one half shown). The arrows indicate in- and out-direction of flow of the fluid in the t h e r m o s t a t i n g jacket. T u n g s t e n wire with its two ends p r o t r u d i n g for W h e a t s t o n e - b r i d g e connection is stretched w i t h i n the capillary. The one end near the stopcock serves also as one electrolysis electrode. The other one is in the second, identical, half of the cell. T h e central o p e n i n g at the left is covered b y the pellet cemented directly to this flat-ground end of t h e cell. Mercury is introduced t h r o u g h the stopcock to fill up the space between the pellet a n d the capillary. The same procedure is applied to prepare the other half of the cell. The m e r c u r y serves s i m u l t a n e o u s l y tor the electrical contacts and as the volume m e a s u r i n g fluid.

Circuitry The d.c. measuring circuitry (Fig. 2a) was composed of a stabilized d.c. source (I) whose output voltage was measured by a precision voltmeter (2). In series with the cell (3) there was a light-spot microammeter (4) and an electronic current integrator (5). Measurements of gas evolution were performed at constant voltage taking readings of current and coulombs. In experiments in which the electrolysis current was held constant it was regulated manually on the stabilized d.c. source (Fig. 2b). The voltage drop over the cell was measured by connecting at certain time intervals a high quality 2/~F condenser in parallel with the cell, and then discharging it over a IM~Q resistor and the current integrator. The voltage could be computed from the readings on the integrator by calibration. In this sense a substitute for a high voltage, high impedance d.c. voltmeter was obtained. ®

®

@

c A

CI

b

c

Fig. 2. The circuitry for m e a s u r e m e n t s at c o n s t a n t voltage (a), at c o n s t a n t c u r r e n t (b), and the bridge for following t h e m o v e m e n t of m e r c u r y inside the capillary of the electrolysis cell (c).

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s. MARI~I~, G. PIFAT, V. PRAVDI6

The menisci of mercury in the capillaries were followed by a simple bridge of the Wheatstone type, taking readings on a Helipot precision potentiometer (Fig. 2c). RESULTS

Most of the measurements were done with a sample of haemoglobin which had adsorbed 18 % H~O. The electrolysis was performed mainly at 35 °, because it was found that in borax 9 proton conductivity ceases sharply below 21°. No measurements began before the position of the mercury in the capillaries was constant on thermostating at a given temperature. When the circuit was switched off the mercury remained stationary and started moving only after switching on again. Finally, the PdC12 test showed well defined black spots which also proved that protons were being discharged at the cathode. There were instances when mercury moved to the anodic side, but hydrogen was demonstrated there too. (This has also been experienced beforeS, 9, and was ascribed to the diffusion of hydrogen through the voids in the pellet.) From the integrator readings, and from the measured mercury displacements the current efficiency (i.e. the measured volume of H 2 divided by the calculated volume of H e from the integrated current) was computed. The results are given in Table I. TABLE I CURRENT EFFICIENCY ( = (VOL. H2)obB./(VOL. I-I2)eale.) FOR ELECTROLYSIS OF HAEMOGLOBIN 2_ 18 % ADSORBED H$O AT 35 ° Current ejficiency (%) Type o[ exp.

Number

Duration (rain) Cathode

C o n s t a n t voltage 15o V C o n s t a n t c u r r e n t 4.1/~A

I 2 I 3 i 4

4175 1635 5 lo 555

73 62 20 94

A node

very small 9 6 18

Total

73 71 26 112

In measurements I and 2 of Table I (constant applied voltage) the current rapidly decreased, but the reproducibility was not good. Nevertheless, the plot (I/current) v s . time was definitely not linear as it should have been according to ROSENBERGs if ordinary electrolysis of water were taking place. In the other two experiments (3 and 4) of Table I the current was kept constant by changing the applied voltage. The time dependence of the voltage change is shown in Fig. 3. Taking into account that for each case a new pellet was prepared the reproducibility is reasonable. B y applying Ohm's law one obtains that ROSENBERG'S hypothesis (ref. 5, Eqn. 6) requires a linear relationship for the data of Fig. 3 which is certainly not the case. Another interesting feature of the latter results (Fig. 3) is the permanent polarization created after some time of electrolysis. For instance, in order to start again with the same current (4.1/,A) 166 V had to be applied after the circuit was switched off for 15 h. The drop in the required voltage even for periods of up to two days was usually less than IO % of the value before switching off. ROSENBERG'S experiments 6 were limited to hydration up to about 7.5 %. On the other hand his equation for the dependence of the energy of activation on hydration B i o c h i m . B i o p h y s . A c t a , 79 (1964) 293-3o~

PROTON CONDUCTIVITY IN SOLID HYDRATED HAEMOGLOBIN

297

is based on relations that are stated to hold only for low coverages. We therefore measured directly the energy of activation for electrical conductivity on a disc of haemoglobin with I8 % adsorbed water, whose thickness was measured before pressing onto it graphite layers which were thinner t h a n usual. Owing to the rapid change in current experienced with time, the circuit was switched on for only I min after each 600[-

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1=const=41 pA 35° C

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17h/6% 15h/38 %

20 mini10 %

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200[-

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=&~oOOoo o I

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I

I

I

5OO

1OOO T i m e (rain)

Fig. 3. The time dependence of applied voltage during constant-current electrolysis of haemoglobin with 18 % adsorbed water. The figures at the arrows indicate the period of interruption of electrolysis with the subsequent percentage depolarization. Run i : squares (open, full, open). Run 2: circles (open, full, open, full). Temperature (°C) 35

30

25

20

i

i

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3.4'

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3'.6

Fig. 4- The Arrhenius plot obtained with 18 % hydrated haemoglobin.

temperature had been stabilised. The temperature stability was checked b y a rapid current measurement after IO min. Starting from - - 3 . 4 ° we obtained the results given in Fig. 4. The least-square calculation for the straight line yields an energy of activation of 0.7 ± 0.2 eV. Solid state electrolysis at 35 ° and 15o V on haemoglobin with only 9.17 % adsorbed water gave negative results: in two experiments lasting 7 and 3 days without Biochim. Biophys. Acta, 79 (1964) 293-3 °o

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s . M A R I ~ I ~ , G. P I F A T , V. PRAVDI~

interruption there was no evolution of gas detectable b y our method of measurement. The same type of experiments on haemoglobin pellets of 40.6 and 46.0 To hydration were positive with current efficiency of 30-80 %. Hydrogen formed rapidly at the cathode, but there was no permanent polarization as encountered for 18 % hydration. In one case (40.6 % hydration) the circuit was switched off after the maintenance of a constant current, 250/,A, raised the applied voltage to 34 ° V. However, a starting voltage of 85 V was sufficient to get the same current again by switching the circuit on after a period of 48 h. An independent measurement with this degree of hydration showed that the polarization disappeared in fact within a few minutes. I t was impossible to prepare a pellet of haemoglobin with 60.0 % adsorbed water owing to the plasticity of the material. The dependence of the dielectric constant on hydration was measured using a WTW-Dekameter. For the present purpose the frequency of IOO cycles/sec was arbitrarily chosen, and the results obtained at 35 ° are given in Table II. The details of the technique will be described later in a paper dealing with the mechanism of molecular motion in this system. TABLE

II

THE DEPENDENCE OF THE DIELECTRIC CONSTANT AND LOSS ON ADSORBED WATER IN THE SYSTEM HAEMOGLOBIN --~ H 2 0 AT 350 AND IOO CYCLES/S H~O(adsorbed %)

~

tg¢5

Remarks

"dry" 9,17 18.oo 40.60 * 46.00

3.0 3.7 4.9 ? ?

0.05 o.o7 o.o9 ? ?

M e a s u r e m e n t s a t i o o c/s i m p o s s i b l e d u e t o l a r g e t g c5

• A t i o o K c ] s : E = 32; t g ~ = 1.8, t h u s q u e s t i o n m a r k s i n d i c a t e v a l u e s s u b s t a n t i a l l y g r e a t e r than these. DISCUSSION

In spite of an apparent complexity of the system we are dealing with, there are certain well established experimental facts which facilitate the evaluation of the present results. PERUTZ and coworkersl°, ~1 gave us a clear picture of the crystal structure of haemoglobin. We are particulary interested here in the size and shape of this globular protein macromolecule. These characteristics were in fact already defined by the same authors at the time when CARDEW AND ELEY 11 studied quantitatively the sorption of water by haemoglobin. The result of the latter investigations was that only about one quarter of the calculated geometrical surface of the haemoglobin molecule is covered by the first monolayer of water molecules, which equals to about 6 % water on a dry haemoglobin basis. PERUTZ12 produced convincing evidence supported by DRABKIN13 et al., that out of 0.82 g liquid water per gram of dry protein in the haemoglobin crystals 0.3 g of water is not available as solvent to the diffusing ions. The author calls it "bound water". The latter findings do not necessarily apply in the present case, because we start from the " d r y " state and add up water b y adsorption. However, it is safe to say, that ROSENBERG'S conclusionsS, s are relevant to a state of hydration in which the B i o c h i m . B i o p h y s . A c t a , 79 (1964) 2 9 3 - 3 o o

PROTON CONDUCTIVITY IN SOLID HYDRATED HAEMO(;LOBIN

2() 0

first adsorption monolayer is hardly completed. Our results for lower content ar~• therefore comparable in this respect to ROSENBERG'S. It would not be surprising at all if at such a low coverage no continuous hydrogen bond network has been formed. We avoid the term "ice-like structure", although the word "continuous" is used in the sense of such a structure, or for that matter, in tile sense of the water chains in lithium sulphate monohydrate (ref. 8)*. The lack of sufficient continuity through hydrogen bonds between water molecules in this case is plausible in view of their being about 6 A apart, as calculated by CARDEW AND ELEY11. The usual o-o distance in hydrogen bonded solids is about 2.7-2.9 A (ref. 14). Thus, it is very likely that proton conductivity, if any, is negligibly small within the first monolayer of adsorbed water molecules. It has been concluded by CARDEW AND ELEVn that a model involving localized adsorption of water molecules on polar side-chains distributed over the outer surface of the haemoglobin molecule is definitely supported by their results. As soon as the first monolayer is formed in this way the newly approaching water molecules are likely to adhere presumably by hydrogen bonds, to those already adsorbed. It has also been established n,'2 that water does not penetrate the haemoglobin molecule. Thus, one can visualize at higher coverages of adsorbed water a build up of a threedimensional hydrogen-bond network, i.e. a hydration shell attached to the haemoglobin molecule. However, a limit should be set to this mechanism by the distribution of the water molecules within the first monolayer, and by the shape of haemoglobin molecules and their mutual arrangement. After the completion of the hydration shell (without sharp boundaries), which requires about 3o °/o of "bound" water 12, additional water can be incorporated in a "liquid" state. We should therefore expect proton conduction to set in at about three to four times the hydration defined by the first monolayer. Our results obtained at 18 ?/o hydration degree leave no doubt that this mechanism of charge and mass transfer is preponderant under the relevant experimental conditions. The results given in Table II support this model. The sharp increase in the dielectric constant after a three-monolayer adsorption is in agreement with TAKASHIMA'S 15 and ROSEN'S16 measurements on globular proteins. In fact, the model proposed here removes any apparent contradiction between the results of adsorption measurements and dielectric measurements. KING AND MEDLEY17 were the first to use electrolysis in the solid state for a study of hydration of fibrilar proteins. Electronic vs. ionic (protonic) conduction has since been frequently discussed, but this simple and yet most direct method seems to have been neglected. In the latter authors' experiment there was no evolution of gas at the anodic side of the cell. Precisely this fact is the strongest evidence for proton conductivity in the solid state. The phenomenon was confirmed earlier (see refs. 8, 9, and the references therein), but usually there is no mention in the literature of what happens at the anode in such experiments. In the present case one might raise the possibility of oxygen being absorbed by haemoglobin if produced at the anode in the course of the ordinary electrolysis of water. However, we worked with methaemoglobin (see above), the irreversibly • The r e a d e r m a y wish to c o n s u l t t h e a r t i c l e b y KLOTZ i n t h e a l r e a d y c i t e d book "Horizons in Biochemistry ''z a n d t h e references in ref. 9 in order to ge t a p i c t u r e of t h e m e c h a n i s m of p r o t o n t r a n s f e r t h r o u g h h y d r o g e n - b o n d chains.

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S. MARI~I~, G. PIFAT, V. PRAVDI(~

oxidized form of (oxy-)haemogiobin (with its iron in the trivalent form), which has no oxygen-absorbing power. The permanent polarization effect is also in support of proton conductivity. That is on discharging a proton from a chain of continuous hydrogen-bonds we actually create a permanent vacancy at the beginning of the given chain, which builds up a polarization-resistance. When there is "liquid" water surrounding the hydration shell of the haemoglobin molecule the decay of the polarization may be due to diffusion of protons from the liquid phase into the hydrogen-bonded network of the "bound" water. More detailed experimental evidence is required before making any quantitative conclusions. The idea that proton migration may be important in transmitting energy (or information) across biological membranes and in particular along biologically important macromolecules has been put forward on several occasions, for instance by RIEHL is and EIGEN 19, but we are not aware of any direct measurements in this respect. The haemoglobin crystals seem to present an exceptional opportunity in that the hydration of the molecule may be studied in great detail through a sequence of solid-gas, solid-quasisolid and solid-quasisolid-liquid interactions. Under suitable experimental conditions the present system may offer a biological model for the non-blocking electrode arrangement devised by EIGEN AND DE MAEYER2°. ACKNOWLEDGEMENTS

This work was financially supported by the Federal Funds for Scientific Research. The authors are indebted to V. MIKULI~I~ of the Forensic Medicine Institute, Zagreb, for advice on the spectrophotometric analysis of haemoglobin. Thanks are due to Dr. D. ROSEN (Chester Beatty Research Institute) for much helpful criticism and for sending us his paper prior to publication. REFERENCES 1 B. PULLMAN AND A. PULLMAN, Nature, 196 (1962) 1137. 2 D. D. ELEY, in M. KASHA AND n. PULLMAN, Horizons in Biochemistry, Academic Press, New York, 1962, p. 341. a j . DUCHESNE, in M. KASHA AND B. PULLMAN, Horizons in Biochemistry, Academic Press, New York, 1962, p. 335. 4 D.D. ELEY AND D. I. SPIVEY, Nature, 188 (196o) 725. 5 B. ROSENBERG, Nature, 193 (1962) 364 . e B. ROSENBERG, J. Chem. Phys., 36 (1962) 816. 7 D. L. DRABKIN, Am. J. Med. Sci., 2o9 (1955) 268. 8 S. MARI~I~, V. PRAVDI~ AND Z. VEKSLI, Croat. Chem. Acta, 33 (1961) 187. 9 S. MARI~I~, V. PRAVDI~ AND Z. VEKSLI, J. Phys. Chem. Solids, 23 (1962) 1651. lO M. F. PERUTZ, M. G. ROSSMANN, A. F. CULLIS, H. MUIRHEAD, G. WILL AND i . C. T. NORTH, Nature, 185 (196o) 416. 11 M. H. CARDEW AND D. D. ELEY, in Fundamental Aspects of the Dehydration of Foodstu~s, Society of Chemical Industry, London, 1958, p. 24. lZ M. F. PERUTZ, Trans. Faraday Soc., 42B (1946) 187. 18 D. L. DRABKIN, A. MAY DYCH, J. RANDALL AND G. GLAUSER, J. Biol. Chem., 185 (195 o) 231. 14 W. FULLER, J. Phys. Chem., 63 (1959) 17o5. 15 S. TAKASHIMA, J. Pol. Sci., 62 (1962) 233. le D. ROSEN, Trans. Faraday Soc., 59 (1963) 2178. 17 G. KING AND J. A. MEDLEY, J. Colloid Sci., 4 (1949) I. 18 N. RIEHL, Naturwiss., 43 (1956) 145; Kolloid Z., 151 (1957) 66. 19 M. EIGEN, in D. HAD~I, Hydrogen Bonding, Pergamon Press Ltd., London, 1959, p. 429. ~o M. EIGEN AND L. DE MAEYER, Proc. Roy. Soc. London, 247A (1958) 5o5. ~1 A. F. CULLIS, H. MUIRHEAD, M. F. PERUTZ, i . G. ROSSMANN AND A. C. T. NORTH, Proc. Roy. Soc. London, A265 (1961) 15 and 161.

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