VOL. 14,1553-1563 (1975)
Adsorption Behavior of Globular Proteins at the Water/Mercury Interface FRIEDER SCHELLER, MICHAEL JANCHEN, and HANS-JORG PRUMKE, Bereich Methodik und Theorie und Bereich Biokatalyse, Zentralinstitut fur Molekularbiologie, Akademie der Wissenschaften der DDR, 1115 Berlin-Buch, Lindenbergerweg 70, DDRI
Synopsis The adsorption of globular proteins a t solidhiquid or liquidhiquid interfaces provides evidence of unfolded molecular conformation. Proteins with high apolar character are strongly unfolded, while those with high polar character are generally incompletely unfolded. Structural changes of globular proteins a t adsorption on mercury electrodes were studied by ac polarography and capacity-time curves. Th e surface area per molecule of nine globular proteins was determined from the adsorption kinetics a t the dropping mercury electrode. For all the proteins investigated, this value was greater than the maximal molecular cross section of the native proteins. The surface area was about 19 Az per amino acid residue, which coincides with the value for unfolded proteins a t the water/air interface. Differences between dropping mercury electrode and hanging drop mercury electrode occurred only with lysozyme and phosphorylase; for the other proteins, the structure of the adsorption layer was independent of the time of interaction at the electrode. Since not all of the reducible groups of the adsorbed proteins come into contact with the electrode, the flattening should be incomplete.
INTRODUCTION The behavior of proteins a t interfaces is important for many biological problems, such as the interaction of proteins with biomembranes and insoluble enzyme carriers, the surface catalysis of reactions (e.g., blood coagulation at plastics1), and the development of ordered structures in the course of evolution. Interface behavior is also important for preparation and analytical techniques,2 such as the purification of enzymes by chromatographic methods, the spray drying of protein^,^ and polarographic and rheological studies on proteins. Beside the oillwater and airlwater interfaces, the mercury electrode is of particular interest for studying the properties of proteins in the boundary layer. Its advantage is that the thermodynamic parameters, surface tension, charge density, surface concentration, and potential, are readily accessible by means of a developed measuring technique. In addition, the field strength in the interface can be varied from zero to about lo4 Vlcm a t constant ionic strength of the solution. Therefore the influence of high electric fields on biopolymers can be investigated. The mercurylwater interface is widely used for the characterization of 1553 0 1975 by John Wiley & Sons, Inc.
SCHELLER, JANCHEN, AND PRUMKE
biological redox reaction^.^-^ In this case, the electron transfer is accomplished by a heterogeneous process between the adsorbed biomacromolecules and the electrode: Mercury electrodes are also used for analytical problems, e.g., the estimation of small amounts of protein? and for the detection of denaturation-renaturation processes.gJ0 Frequently, it is assumed that proteins have identical structure in the solution and in the adsorption layer of the e l e ~ t r o d e . ~ , ~However, Jl it is known that proteins undergo structural changes at the interface. Evidence for such structural changes of adsorbed proteins was obtained by Kuznetsov12 from the BrdiEka polarographic catalytic currents13 of seven globular proteins, as well as from capacitance-time curves at the hanging drop mercury electrode (HDME). Kuznetsov stated that during adsorption, an irreversible unfolding occurs; this finding is of fundamental importance for electrochemical studies of proteins. In the present work, studies are reported on the adsorption kinetics of nine globular proteins. From these measurements, the area that is occupied by one of the adsorbed molecules at the electrode was determined. This value is compared with the surface area in the waterlair interface and with the cross section of the molecule. In contrast to Kuznetsov's results, our studies consider the interaction times between mercury and proteins from 3 to 25 sec. Investigations of the adsorption kinetics were performed by means of ac polarography and capacitancetime curves a t constant potential. EXPERIMENTAL Methods The ac polarograms and the capacitance-time curves were recorded by a polarograph GWP 563 (Zentrum fur Wissenschaftlichen Geratebau der AdW der DDR, Berlin-Adlershof). The ac frequency of this instrument is 78 Hz; the amplitude was adjusted to 10 mV. In the potential region of maximal decrease of capacity,. the measured alternating current is proportional to the differential capacitance of the e1ectr0de.l~ The recorder reading was checked by a calibration condenser. The long drop-time electrode, according to Smith,15 has a resistance of 50 f2 and allows drop times up to 30 sec. For ac polarography, capillaries with a 0.1-mm internal diameter and 15-cm length were used. The potentials were related to the saturated calomel electrode, which we used as counterelectrode (KE 15, Forschungsinstitut Meinsberg). The temperature of the vessel was maintained a t 2 5 O f 0.1OC. The solution volume was varied from 2 to 20 ml.
Materials The background solution was prepared from twice recrystallized KC1 in doubly distilled water. The pH of the protein solutions was adjusted
GLOBULAR PROTEINS AND H20/Hg
to 7.0 by the addition of 0.1 M HC1 or 0.1 M KOH, insulin was measured at pH 3. Dissolved oxygen was removed by bubbling the solution with purified nitrogen. Insulin, lysozyme, ribonuclease (RNAse), and horse heart cytochrome c were purchased from Reanal, Budapest. Egg albumin and bovine serum albumin (BSA) were obtained from VEB Serumwerk Dessau. Bovine metmyoglobin (MetMb), human methemoglobin (MetHb), and rabbit muscle glycogen phosphorylase b were prepared by standard methods,16J7 and were separated from other electrolytes by gel filtration across a Sephadex column loaded with 0.1 M KC1 solution.
Evaluation of Experimental Data For strongly surface-active substances, any molecule reaching the surface is instantaneously adsorbed, and desorption is negligible.14J8 In this case of diffusion-controlled adsorption, the Koryta equationlg describes the dependence of the surface concentration, r on time t and concentration c in the soliltion:
r = 7.36 x 10-4.~1/2.~.t1/2
where D is the diffusion coefficient. Provided that 1) the relative decrease of capacitance ACICO is proportional to the degree of coverage of the electrode,202) that the surface area S occupied by the protein molecule in the surface does not depend on the degree of coverage21and 3) that only one monolayer is formed,21 one can formulate the following relationship for the capacitance-time curve:
*NA *S-D1’2*C*t’’2 .ACs/Co
where Cs corresponds to the full coverage of the electrode. The surface area of a molecule is also obtained from the ac polarograms representing capacitance average values. According to Jehring and Horn,21 integration over the drop time is taken into consideration by introducing the factor 0.77 in Eq. (2): - -
0.77 X 7.36 X
_ _ .N,.S.D”2.c.t,”2.AC,/Co
where t, is the drop time.
RESULTS The ac polarograms (Figures 1 and 2) of the protein solutions show a sharp decrease of capacitance in comparison with the background solution near the zero charge potential (adsorption region). The capacitance decreases with increasing protein concentrations, reaching a saturation value, which corresponds to a completely protein covered electrode (Figure 1). For RNAse, lysozyme, egg albumin, and BSA, the ac
SCHELLER, JANCHEN, AND PRUMKE
Fig. 1. Ac polarograms of insulin in 0.1 M KCl, pH 3. I-background solution, 2g/l., 4-0.067 g/l., 5-0.100 g/L, 6-0.200 g/l., 7-0.400 g/l.
0.025 g/l., 3-0.050
Fig. 2. Ac polarograms of BSA in 0.1 M KCl. I-background solution, pH 7 , 2-0.13 PA., 3-0.22 g/l., 4-0.65 g/l., 5-1.50 g/l.
polarograms show, in addition to a cathodic desorption peak, a second peak a t about -800 mV, which is ascribed to the rearrangement of adsorbed moleculeszz or to the reversible reduction of disulfide bridgesz3 (Figure 2). On the other hand, insulin, hemoproteins, and phosphorylase did not yield any peaks in the adsorption region (Figure 1). The relative lowering of the capacitance at constant potential in the adsorption region depends linearly both on c and t m 1 f Z(Figure 3) for
GLOBULAR PROTEINS AND H20/Hg
05 0.4 .
0.3 0.2 . 0.1 .
the proteins investigated. The diffusion is the rate-determining step for the adsorption process. The surface area of an adsorbed protein molecule was calculated using Eq. (3). The values determined in this manner are listed in Table I. The shape of capacitance-time curves at constant potential of the long drop-time electrode is typical of strongly surface-active substances21 (Figure 4a). The lowering of capacity in the adsorption area increases with rising protein concentrations up to a saturation value. The AC/Co versus t1/2 plot of these curves yields straight lines. A t high concentrations, time-independent saturation occurs (Figure 4b). The slope of the straight lines is proportional to the protein concentration. This shows the validity of Eq. (2), i.e., that the adsorption of the proteins is limited by diffusion. The values of the surface area determined for six proteins are listed in Table I. Deviations from the linear progression of the AC/Co-t1/2 curves occurred a t concentrations above 0.02 gll. with RNAse, lysozyme, egg albumin, and BSA. It may be assumed that, by analogy with the concentration dependence of the ac polarograms (Figure 2), the peak shifted towards positive potentials at high surface concentrations.
DISCUSSION The S values determined from the concentration dependence of the C-t curves coincide with those obtained from the concentration or droptime dependence of the ac polarograms within 20% (Table I). Only the values of the C-t curves for RNAse and lysozyme are considerably higher. Our value obtained from the ac polarograms corresponds to the sur-
SCHELLER, JANCHEN, AND PRUMKE
Fig. 4. (a) C-t curves of metmyoglobin, -700 mV. (b) Variation of the relative decrease of differential capacity, AC/Co with drop age. 1-0.005 gh., 2-0.0075 g h . , 30.010 g/l., 4-0.020 g/l., 5-0.0400 gh., 6-0.1 M KC1.
face area of 1800-2500 Az per RNAse molcule determined by PavloviE and Millerz3using a capacitance bridge and the DME. The evidence of the S values obtained by us is also shown by the accordance with results of dc polarographic studies on insulin, RNAse, and BSA.33 1) With these proteins, the limiting current of the reduction of disulfide bridges rises linearly with concentration, reaching a maximal value a t high concentrations. The intercept of the two straight lines gives the bulk concentration a t which the mercury surface is just fully covered a t the given drop time. The saturation surface concentration and its reciprocal, the area S, are calculated from this value with the aid of Eq. (1) (Table I). The S values of insulin and RNAse do not differ a t solutions with different pH (pH 1 and pH 7.1). 2) It may be suggested that a t maximal current an adsorbed protein monolayer is reduced with each mercury drop. This fact offers one the
GLOBULAR PROTEINS A N D H2O/Hg
TABLE I Surface Areas of Globular Proteins Surface Area S (A2/molecule) Interface Mercury/Water
Insulin Cytochrome c RNAse
d.c. polaroAir/Water gram 87030 -
area S* ness (A* /re%) d b ( A )
( E = -800 m V ) 2.100 2,200 ( E = -700 m V )
( E = -600 mV)
MetHb (dimer) Ovalbumin
( E = -600 m V )
BSA Glycogen phosphorvlase b a
9,8003' 11,0009,20033 13,0003* -
8,500 3,600 ( E = -700 m V ) 5,700 ( E = -600 m V ) 7,300 6,400 ( E = -700 m V ) 10,100 13,000 ( E = -700 mV) 7,300 ( E = -1,000 mV) ~
Greatest surface of the rectangular prism, which completely covers the molecular model. X S X 1.3 (A).
b d = M/0.6
opportunity to calculate the saturation surface concentration from the drop surface, the number of SS groups reduced per molecule, and the charge i-t,. Here, we did not use the precondition for evaluating the adsorption kinetics, Eq. (1). These values coincide with those obtained by Eq. (1) within 15%. The surface area allows one to draw conclusions about the structure of the adsorbed molecules. The electrode surface area S of the seven proteins for which comparative values are available is higher than the biggest cross section of the molecule in the crystalline state. Two reasons may be offered to explain this finding. 1) It is well known that globular proteins may unfold under the influence of interface energy. The degree of unfolding should be consistent with the maximal lowering of the free surface energy. The electric field across the interface may also induce unfolding.34 2) Repulsion forces between the adsorbed molecules may cause the proteins 'to form only a loose layer a t the electrode. At the water/air interface, Birdi and F a ~ m a nand ~ ~Birdi36,37determined a value of about 17 A2 per amino acid residue for different globular proteins from limiting slopes of the two-dimensional state equation. This value corresponds roughly to the surface occupied by one amino acid of the @-sheetstructure,38 as established by X-ray studies. From this finding and the high isothermic work of compression with insulin, Hb, egg albumin, and BSA, Birdi concluded that proteins with high apolar character are completely unfolded a t the water/air interface. Table
SCHELLER, JANCHEN, AND PRUMKE
I shows the excellent coincidence for these proteins a t the mercury/ water and airlwater interface. In contrast, proteins with a high polar character (e.g. lysozyme, cytochrome c, Mb) are partially unfolded at the waterlair interface and the area per residue is about 4 A2. Kuznetsov,12 investigating the BrdiEka polarographic catalytic current of seven globular proteins, has established that the value of wave I divided by the surface concentration of the sulfur atoms of the S H and SS protein groups is (0.37-0.57) X A. He concluded that all the mercapto and disulfide groups of these proteins are accessible for the electrode process. He assumes that the participation of groups from the interior of the molecule is due to complete unfolding of globular proteins on adsorption a t the HDME. He calculated a thickness d of the adsorbed layer of 4-6 A from the concentration dependence of the first catalytic wave and of 5-7 8, from the second wave. Kuznetsov12 obtained a thickness of 8 A for BSA from the C-t curves at the HDME according to Eq. (1). He concluded from these values that the layer of adsorbed proteins has the thickness of a polypeptide chain. The calculation of the thickness is based on the assumption that the protein density is the same as in the crystal and that the film is of uniform thickness. But this value is an average over the entire adsorption layer. An analogous result was obtained by Nurnberg and coworkers39 who studied the electrochemical behavior of native DNA. They inferred a helix-coil transition of adsorbed DNA induced by the electric field at the interface, because the cytosine and adenine bases become accessible for the electrode reaction. Table I shows that the ac polarographically determined d values coincide well with those obtained by Kuznetsov12 from the second catalytic wave in BrdiEka's solution and from the C-t curves, except for those of lysozyme and phosphorylase. This coincidence suggests that the unfolding of these proteins on adsorption proceeds rapidly. A t the DME, long drop-time electrode, and HDME, the protein adsorption layers have approximately the same structure. For RNAse, lysozyme, and phosphorylase, the differences of S values at the DME and HDME may indicate structural changes occurring several seconds after incorporation into the interface. The S* values per amino acid residue obtained by us with the DME are in the range from 13.5 to 23.5 A2. These differences between the individual proteins may indicate a different extent of unfolding. However, this effect may also be due to the differences in primary structure, i.e., if the structure in the adsorption layer is almost uniform, the average area per amino acid is determined by the dimensions of the individual amino acids. Figure 5 shows that a linear relationship exists between the average amino acid residue volume V40 and the S* values. Using a computer we calculated a correlation coefficient of 0.71 for the relationship S* = 0.23 V. This result suggests a greatly uniform structure of the adsorbed proteins.
GLOBULAR PROTEINS AND H20/Hg
. . -.------.
Fig. 5. Dependence of surface area S* at the mercury electrode on the average amino acid residue volume V for eight globular proteins.
The studies by Kalous41 and MullerlO in BrdiEka's solution suggest that in the adsorption layers, not all SH or S S groups are accessible for the electrode (polarographically buried groups). The denaturation of globular proteins by urea, guanidine hydrochloride, or alkali causes an increase of the wave height compared with native proteins. This is due to the increase in the number of S atoms that are accessible for the electrode. Cecil and W e i t ~ m a nPavloviE ,~~ and Miller,23and Freimuth and c o - ~ o r k e r shave ~ ~ found that only part of the SS groups of insulin, RNAse, BSA, trypsin, and P-lactoglobin is cathodically reducible. These results show that during the DMEIprotein interaction (about 3 sec), globular proteins are not completely flattened at the interface. In analogy, the adsorption at the waterlair interface is not equivalent to total destruction of secondary ~ t r u c t u r e . ~The ~ , ~degree ~ of helicity of protein and polypeptide monolayers is only slightly affected by the Thus monolayers of ovalbumin with a thickness of 8 A, which agrees with the value at the mercury electrode (Table I), can be further expanded by ultraviolet irradiation or heat denaturation of the protein.46 The limiting area per residue in a helical structure is not significantly different from that in the P - s t r ~ c t u r ethat ; ~ ~ is why the value of 17 A2 is no proof of the degree of unfolding. The results at the mercury electrode give evidence that, in the adsorption layer, the globular proteins are unfolded and that in these layers helical structure and intrachain bonds remain intact to some extent.
SCHELLER, JANCHEN, AND PRUMKE
In contrast, the alteration of proteins in the solution may yield adsorbed molecules, which are completely unfolded. For example Kolthoff and c o - ~ o r k e r found s ~ ~ that in the presence of calcium chloride, all disulfide groups in BSA are reducible at the electrode. Phosphorylase, for which the layer thickness is much higher than for the other proteins, evidently undergoes small changes on adsorption. I t is generally expected that proteins that act at the interface are especially resistent to unfolding by interfacial energy.49 A t high concentrations, a rapid saturation of the interface can prevent irreversible structural changes of the adsorbed proteins. Therefore, it is possible to obtain native reduction products by the cathodic reduction of protein^.^^^^ The authors wish to thank Dr. G. Etzold and Dr. M. Falck for helpful discussions and Mrs. I. Seyer for excellent technical assistance.
References 1. Lyman, D. (1974) Angew. Chem. 86,145-150. 2. Mac Ritchie, F. (1972) J. Colloid Interface Sci. 38,484-488. 3. Trouwborst, T., De Jong, J. & Winkler, K. (1973) J. Colloid Interface Sci. 45, 198208. 4. Theorell, H. (1938) Biochem. Z . 298,25%260. 5. Weitzman, P., Kennedy, J. & Caldwell, R. (1971) FEBS Lett. 17,241-244. 6. Betso, S., Kalpper, M. & Anderson, L. (1972) J. Amer. Chem. Soc. 94,8197-8210. 7. Berg, H., Granath, K. & Nygard, B. (1972) Electroanal. Chem. 36,167-178. 8. PaleEek, E. & Pechan, Z. (1971) Anal. Biochem. 42,59-71. 9. Ruttkay-Nedecky, G. & Bezuch, B. (1971) Exp. Suppl. 18,553-562. 10. Muller, 0. (1963) in Methods of Biochemical Analysis, Glich, D. Ed. Wiley, New York, Vol. 11,pp. 329-403. 11. Behr, B., Bialowolska, M. & Chodkowski, J. (1973) J. Electroanal. Chem. 46, 223231. 12. Kuznetsov, B. (1971) Exp. Suppl. 18,381-386. 13. BrdiEka, R. (1933) Collect. Czech. Chem. Commun. 5,122-128. 14. Jehring, H. (1969) J. Electroanal. Chem. 21, 77-98. 15. Smith, G. (1949) Nature 163,290-292. 16. Fischer, E. & Krebs E. (1962) in Methods in Enzymology, Colowiek, S. & Kaplan, N. Eds., Academic, New York, Vol. 5, pp. 369-372. 17. Theorell, H. (1932) Biochem. 2. 252,l-8. 18. PavloviE, 0. & Miller, J . (1971) J. Polym. Sci., p t . C 34, 181-200. 19. Koryta, J. (1953) Collect. Czech. Chem. Commun. 18,206-209. 20. Damaskin, B., Petry, 0. & Batrakov, V. (1968) in Adsorption of Organic Substances at Electrodes, Nauka, Moscow, pp. 55-109. 21. Jehring, H. & Horn, E. (1968) Monatsber. Deut. Akad. Wiss. Berlin 10,295-306. 22. Berg, H. (1966) Abh. Deut. Akad. Wiss. Berlin, Klasse Medizin 1966, 479-484. 23. PavloviE, 0. & Miller, J. (1971) E r p . Suppl. 18,513-524. 24. Adams, M., Baker, E., Blundell, T., Harding, M., Dodson, E. et al. (1969) Nature 224,491-495. 25. .Dickerson, R., Takaro, T. & Eisenberg, D. (1971), J. Biol. Chem. 246,1511-1533. 26. Kartha, G., Bello, J. & Harker, D. (1967) Nature 213,862-865. 27. Krighaum, W. & Kugler, F. (1970) Biochemistry 9,1216-1223. 28. Damaschun, G. (private communication).
GLOBULAR PROTEINS AND H20/Hg
29. Fasold, H., Orttanderl, F., Huber, R. & Bartels, K. (1972) FEBS Lett. 21,229-232. 30. Birdi, K. (1972) presented a t the 6th Int. Congr. Surface Active Substances, Zurich. 31. Khaiat, A. & Miller, J. (1969) Biochim. Biophys. Acta 183,309-319. 32. Birdi, K., Gabrielli, G. & Puggelli, M. (1972) Kolloid 2. 2. Polym. 250,591-593. 33. Cecil, R. & Weitzman, P. (1964) Biochem. J. 93,l-11. 34. Bean, C. & Bennett A. (1973) Biopolymers 12.817-824. 35. Birdi, K. & Fasman, G. (1972) J. Polym. Sci., p t . A 10,2483-2486. 36. Birdi, K. (1972) Kolloid 2. 2. Polym. 250,222-226. 37. Birdi, K. (1973) J . Colloid Interface Sci. 43,545-547. 38. Malcolm, B. (1968) Proc. Roy. Soc., Ser. A 305,363-365. 39. Valenta, P., Nurnberg, H. & Klahre, P. (1974) Bioelectrochemistry 1,487-505. 40. Bull, H. & Breese, K. (1973) Arch. Biochem. Biophys. 158,681-686. 41. Kalous, V. (1971) Exp. Suppl. 18,349-354. 42. Notzold, H., Schlegel, B., Tinius, J. & Freimuth, U. (1972) 2. Chem. 12,24-25. 43. Brash, J. & Lyman, D. (1971) in The Chemistry of Biosurfaces, Hair, M. Ed., Dekker, New York, Vol. 1, pp. 177-229. 44. Inbar, L. & Miller, J. (1974) Biochim. Biophys. Acta 364.146158. 45. Loeb, G. & Baier, R. (1968) J . Colloid Interface Sci. 27,38-45. 46. Kaplan, J. & Fraser, M. (1953) Nature 171,559-560. 47. Miller, J. & Bach, D. (1973) in Surface and Colloid Sci., Matijevic, E., Ed., Wiley, New York, Vol. 6, pp. 186255. 48. Kolthoff, J. Yamashita, K. & Boen Hien, T. (1974) Proc. Nat. Acad. Sci. US.71, 2072-2076. 49. Brockerhoff, H. (1974) Chem. Phys. Lipids 10,215-222. 50. Scheller, F. & Janchen, M. (1974) Stud. Biophys. 46,153-157.
Received September 10,1974 Accepted March 12,1975