Protein Response of Silver Sulfide Crystal Membrane Electrodes Paul D’Orario and G. A. Rechnitz” Department of Chemistry, State University of New York, Buffalo, N. Y 14214
The direct response of Ag2S crystal membrane electrodes to sulfur-containing proteins has been Investigatedusing model compounds of varying disulfide and sulfhydryl content. Potentiometric measurementson solutions of the proteins lysozyme, ovalbumin, human serum albumin, and ribonuclease A show clear relationships between electrode response and protein conformation. Studies involving denaturation and refolding of model proteins demonstrate that electrode response is a function of the free sulfhydryl content of proteins and not of disulfide linkages. The mechanistic Information obtained lays the groundwork for the analytical use of Ag2S electrodes for the direct measurement of proteins in solution.
Recently, ion selective electrodes have found increasing use in biological media and as tools for clinical analysis (1).Such studies have included the use of the solid state silver sulfide membrane electrode for both direct and indirect determination of organic and biological species (2-4). Thiols have been determined directly (5), as well as by titration (6-8) and the argentometric titration of proteins has been reported (9-11). The basis of such titrations is the interaction of silver ions with the sulfhydryl groups of the cysteine residues in proteins. This interaction has been shown t o be quite strong ( 5 ) and is thought t o be the predominant binding site for silver on a protein molecule (12). Many protein titration studies have given erroneously high results which may indicate either unspecific binding of silver ions t o sites on protein molecules or a silver binding in excess of the expected 1:l Ag-SH ratio (l3--16).Although these deviations have partly been eliminated by titrations in imidazole rather than the classical Tris or ammonia buffers ( 7 , 1 7 ) ,several sites on protein molecules, including the amino acids lysine, arginine, and methionine have been shown capable of binding silver. However, it is believed that these interactions cannot account for excess silver binding at the concentrations normally employed in protein titrations (6). T h e purpose of this work is to investigate the potentiometric response of the silver sulfide crystal membrane electrode to proteins directly, e.g., without the addition of metal ion reagents. Mechanistically, such a response would occur via the interaction of the protein sulfhydryl groups with the minute amount of silver ions always present a t the silver sulfide-solution interface. In order t o elucidate the nature of the protein response and to attempt some quantitative correlation, model proteins were chosen based on their disulfide and sulfhydryl contents. T o separate the variables, standard methods of protein treatment including denaturation as well as reduction and reoxidation of disulfide bonds have been employed. Hopefully, such electrode parameters as response slope and detection limits can then be discussed and compared for the various proteins. During the course of this work, it was demonstrated that electrodes can also be useful for basic studies of protein conformation and structure. Direct potentiometric monitoring of proteins using ion selective electrodes may have applicability in clinical situations. It is therefore important t h a t the fundamental processes of electrode response t o proteins be more thoroughly understood
before the practical applications of electrodes to such measurements be attempted.
EXPERIMENTAL Apparatus. All potentiometric measurements were made with a Corning Model 12 Research pH meter in conjunction with a HeathSchlumberger Model SR-255B Strip Chart Recorder. The indicator electrode was an Orion Model 94-16A silver sulfide membrane electrode used with an Orion Model 90-01-00 single junction reference electrode. Electrical contact between the indicator and reference electrodes was made via a 10% KN03 agar salt bridge. The sample cell used was a 10-ml Teflon minibeaker which was specially modified when the exclusion of air from the sample solution was necessary. All potentiometric measurements were made at room temperature. Protein separations were carried out routinely on a 2.5 X 25 cm glass column which had been previously packed and equilibrated. Fractions were collected using a Technicon AutoAnalyzer Sampler. All spectrophotometric measurements were carried out on a Beckman DB-G Grating Spectrophotometer. pH determinations were made with a Corning semi-micro, combination pH electrode. Reagents. The proteins used were obtained from the following commercial sources: ribonuclease A (lyophylized, Type I-A from Bovine Pancreas), ovalbumin (lyophylized, Grade VI, 99% pure by electrophoresis), and human serum albumin (crystallized and lyophylized) were obtained from Sigma Chemical Co., St. Louis, Mo. Lysozyme (3X crystallized and lyophylized) was obtained from ICN Life Sciences, Plainview, N.Y. Dithiothreitol (Cleland’s Reagent), 5,5’-dithiobis(2-nitrobenzoicacid) (Ellman’s Reagent), L-cysteine (free base), and Sephadex G-25-300beads for gel filtration were also obtained from Sigma Chemical Co. Ultrapure urea was purchased from Schwarz-Mann Biochemical Co. Orangeburg, N.Y. All buffers were prepared from standard tables (18)using analytical grade reagents and were 0.9% in saline at a constant ionic strength of 0.16 M. These included:*aceticacid-sodium acetate (pH 4.01, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 6.0 and 7.0) and boric acid-sodium borate (pH 8.5). Tris-hydrochloric acid buffer (pH 8.0,I = 0.1 M) was prepared and used for the Ellman colorimetric test. Other analytical grade reagents used throughout the course of this work include: silver nitrate, disodium EDTA, sodium hydroxide, and sodium chloride. Procedures. Stock protein solutions were prepared daily and in sufficiently low concentrations to prevent protein aggregation, especially for the albumins. The lyophylized powder was dissolved directly in the appropriately buffered saline in concentrations ranging from 1.0 t o 2.5 mg/ml. This range was found to be convenient inasmuch as potentiometric measurements were carried out at significantly lower protein concentrations by increment addition of the stock solution to constant volumes of the various buffers. Stock solutions could also be prepared, at these low concentrations, in the vicinity of the isoelectric pH’s for individual proteins. Potentiometric studies of native proteins were carried out in the following manner. The electrodes were immersed in buffered saline solution until a stable, reproducible potential was obtained. The electrodes were then dipped into similarly buffered solutions of increasing protein concentration and the resulting potentials compared to the previous blank potential. Upon reimmersion of the electrodes into a blank, the potential was found not to reproduce the original blank but to be dependent on previous exposure to protein, becoming progressivelymore negative as time of exposure to protein and protein concentration increased. This can be attributed to the well known fact that solid-state membrane electrodes are susceptible to protein poisoning (9).It was observed that reproducible blanks could be obtained if the AgjS electrode was washed in M silver nitrate for several minutes following each blank and protein measurement. Nevertheless some drift of the blank potential upon continuous use of the electrode in protein solutions was unavoidable and, therefore, potential measurements are presented as AE values, that is E B l a n k - Eprc,te,n. The silver sulfide membrane electrode was observed to acquire a dullness on the sensing surface upon prolonged use. Therefore the membrane ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
41
Table I. Model Proteins Studied" Protein
Disulfide content (mol S-S/mol protein)
Sulfhydryl content (mol SH/mol protein)
Mol w t
4
0 3-4 0.7 0
14 800 46 000 66 000 13 700
Lysozyme Ovalbumin Human serum albumin Ribonuclease A a
1 17 4
Data from Ref. 25.
20-
z W 4
02
04 Plotel"
C0"C
06 mg/m1
08
Figure 1. AgPS membrane electrode response to the native proteins lysozyme (A) and ribonuclease A (B) at pH 8.5
D
70
Ovalbumin
C O ~ C . ,m g i m l
Figure 2. AglS membrane electrode response to the native protein ovalbumin as a function of pH (A) pH 4, (6) pH 6, (C)pH 7, and (D) pH 8.5
was periodically polished to a mirror-like finish with Diamantine diamond powder of decreasing grades of coarseness. After polishing, it was necessary to recondition the electrode in a concentrated protein M silver nitrate before resolution followed by conditioning in producible blanks could once again be established. It is known that trace concentrations of metal ions are responsible for the catalysis of the oxidation of sulfhydryls to disulfides (12). Therefore protein reduction and separation was performed in glass42
02
OB
14
10
HSA
conc
18
I
rngirnl
Figure 3. Ag& membrane electrode response to the native protein human serum albumin at pH 8.5
ware which had been rinsed with M EDTA. Protein was dissolved and denatured in 8 M urea and reduced with excess quantities of 10-l M dithiothreitol (DTT) prepared by dissolving the appropriate amount of solid in deionized water. Fresh solutions of DTT were prepared prior to use. The protein treatment was allowed to proceed with stirring for several hours. Separation of the reduced protein from the denaturation media was performed by gel filtration on a Sephadex G-25 column, equilibrated with acetate buffered saline at pH 4. Fractions were collected at a flow rate of 4 ml/min and checked for SH activity using the Ellman colorimetric test. The Ellman reagent was prepared in 5 X IO-" M concentration by dissolving an appropriate amount of solid in pH 8.5 borate buffered saline. The reagent's extinction coefficient was determined to be 10 300 M-' cm-' by calibration with cysteine and optical measurements were made at 412 nm as described in the literature (19).The appropriate fractions were pooled and diluted to known volume with acetate buffered saline. Reoxidation was performed by raising the pH of the stock solution to between 8 and 9 with concentrated NaOH and exposing the solution to oxygen (20, 21). After variable periods of time, samples were withdrawn from the stock solution and spectrophotometric and potentiometric measurements were done simultaneously. The negative logarithm of sulfhydryl concentration was then plotted vs. electrode potential at constant protein concentration. It should be noted that potentiometric measurements of the reduced protein were carried out in borate buffered saline (pH 8.5) which had been previously purged with nitrogen and stored under air-tight conditions. Similarly, nitrogen was forced through the sample cell to minimize the effect of air oxidation of the reduced protein during the electrode measurements.
_I / I
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
RESULTS AND DISCUSSION T h e Ag2S crystal membrane electrode responds t o inorganic S2- via an equilibrium change in Agf activity at the membrane surface ( 2 2 ) ;as a result, t h e response slope for S2- is one-half that observed for Ag+ and t h e direction of t h e potential change is reversed. When the Ag2S electrode is employed to measure sulfur-containing organic or biological molecules, it is reasonable to suppose that similar interactions between sulfur -containing functional groups and Ag+ may be involved, although the actual response slope would depend upon t h e stoichiometry of the interaction. On theoretical grounds ( 2 3 ) ,response slopes as large as
Figure 4. Separation profile of reduced ribonuclease A from reagents. Each fraction contains 4 mi of effluent
Figure 5. AgPS membrane electrode response to reduced ribonuclease A at pH 8.5
-118 mV per decade concentration could be expected for materials forming 1:2 silver-ligand complexes. Experiments on low molecular weight model compounds such as cysteine show slopes approaching this theoretical limit. Tseng and Gutknecht ( 5 ) ,who obtained a slope of -108 mV/decade of cysteine concentration, reasonably propose the formation of a Ag(-SCHa C H ( N H Z ) C O ~ - ) ~complex ~with a calculated formation constant of 3.2 X 1013a t an ionic strength of 0.1 M. These authors found linear calibration curves only t o M, however, whereas our measurements show linearity as low as 5X M. This concentration is considerably lower than the limit expected on the basis of the lattice defect hypothesis (23) but is consistent with the view t h a t sensitivity limits are governed by solubility of the electrode membrane (24). Response to Proteins. The situation with regard to high molecular weight proteins can be expected to be considerably more complicated. First, proteins will have several binding sites per molecule and, perhaps, several types of functional groups a t these sites. Second, not all of the possible binding sites will be accessible when the protein is in its native, folded state. Third, the p H dependence of binding is likely to be complicated owing t o differing pK’s of the various binding sites. In order t o separate the variables and elucidate the nature of protein response a t Ag2S electrodes, we selected the model proteins listed in Table I for systematic study. I t should be noted t h a t these proteins have a range of molecular weights and of number of sulfur-containing functional groups. Moreover, while all four proteins contain disulfide linkages in the native state, neither lysozyme nor ribonuclease A have any sulfhydryl groups in the folded state. N a t i v e (Folded) Proteins. Figures 1-3 show the direct response of the Ag2S electrode t o increasing concentrations of the four proteins. First, it should be noted t h a t the two proteins which have no sulfhydryl groups in the native state, Le., lysozyme and ribonuclease A, give little potential change with increasing protein concentration (Figure 1).This finding is in agreement with our hypothesis t h a t the Ag2S electrode responds only t o the free sulfhydryl content and not the disulfide content of the protein. The small potential changes observed are most likely residual effects from slow alkaline denaturation (unfolding) a t p H 8.5 since only negligible potential changes are observed a t lower pH. Any unfolding of the native proteins would tend t o rupture disulfide bonds. The lack of response to lysozyme and ribonuclease A can be contrasted with the large potential changes observed for increasing concentrations of ovalbumin which are shown in Figure 2 as a function of pH. As expected (25) the response is sharply p H dependent with greatest response observed
when the p K of the sulfhydryl groups (thought to be approximately 8.3 (26))is exceeded and the binding sites become negatively charged. Comparison of the response at pH 8.5for ovalbumin (Figure 2) and human serum albumin (Figure 3) lends further support to the sulfhydryl response hypothesis. As shown in Table I, human serum albumin contains 17 times more disulfide linkages than ovalbumin, yet electrode response to ovalbumin is considerably greater than to human serum albumin in accord with the relative sulfhydryl content of the respective proteins. Ribonuclease-A Model System. Unambiguous confirmation of our hypothesis can be obtained from studies on ribonuclease A because this protein, which has no sulfhydryl groups in its native state, can be quantitatively denatured with reduction of disulfide linkages to sulfhydryl groups and subsequently reoxidized to reverse the conversion. Denaturing agents, such as 8 M urea (27),and various reducing agents can be used to expose and cleave the disulfide bonds which stabilize the tertiary structure of native proteins. Recently, dithiothreitol has become widely accepted (28) as a reducing agent for this purpose. If sulfhydryl groups do indeed determine potentiometric response, then the conversion of disulfide linkages to sulfhydryl groups should produce large changes in electrode response a t relatively low protein concentrations. Ribonuclease A was, therefore, denatured and reduced using the method of Anfinsen and Haber (20) with minor modifications. For subsequent electrode measurements, it is important that all possible interferants be removed after the chemical conversion of the protein. Since both residual urea (10) and the reducing agent, being a thiol, would in themselves produce some electrode response, the denatured protein was separated from other reagents and products by the method of Anfinsen and Haber. T h e separation was carried out on a column conditioned with acetate buffered saline solution at, pH 4 where reoxidation of sulfhydryl groups is prevented even under less than perfectly air free conditions. A typical profile of the separation is shown in Figure 4 and testing (29) of the protein fractions for urea showed t h a t this interferant had been reduced to negligible levels. Fully reduced ribonuclease A should contain 8 sulfhydryl groups per molecule; we routinely found values in excess of 7.9 for our preparation and further found changes of no more than 1-2% relative in sulfhydryl content from day to day storage a t p H 4.This enabled us to obtain calibration curves for reduced ribonuclease A with negligible changes in protein sulfhydryl content of the stock solutions during the course of t h e measurements. Typical of our results is Figure 5 in which -log [SH] is plotted vs. potential change. The linear portion of this curve exhibits a near ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
43
Table 11. Detection Limits of the AgzS Electrode for Protein Sulfhydryl Content Protein
-log SH concn
OA HSA RNase A (reduced)
5.3 5.5 5.3-5.5
Apparent detection limit SH concn, M 5.0 X 3.2 X 3.2 X 10-6-5.0 X
Corresponding protein concn, mg/ml 0.05 0.3 0.005-0.008 a
Assuming complete reduction of the RNase A disulfide bridges is achieved.
a
Analytical Implications. The results of these studies suggest that AgzS membrane electrodes could be used for the direct measurement of sulfur-containing proteins without addition of metal ions. Further, it is clear that qualitative distinctions could be made among individual proteins on the basis of their sulfhydryl content or by making measurements before and after denaturation. Some preliminary detection limits, based upon the sulfhydryl content of the proteins studied are given in Table 11. LITERATURE CITED
I
I 52
54
56 - L o g SH conc
56
80
M
Figure 6. Ag2S membrane electrode response to the reoxidation of reduced ribonuclease A at pH 8.5. Protein concentration is constant at 0.01 mg/ml
Nernstian response slope of 56.5 mV per decade over the concentration range shown. This would indicate that a 1:lAg+ to SH stoichiometry is involved in the potential determining equilibrium a t the electrode surface. The reoxidation of reduced ribonuclease A is easily carried out using well established methods (21,30). Previous work (21) indicates that the original conformation and enzymatic activity are restored during oxidation; thus, the reduction and reoxidation of ribonuclease A can be regarded as a fully reversible cycle. This is an attractive property of the model system for purposes of the present work because it permits us to monitor the disappearance of the sulfhydryl groups formed by reduction of the disulfide linkages as the ribonuclease A is being reoxidized. Such an experiment, involving simultaneous potentiometric and spectrophotometric measurements, is shown in Figure 6 for a constant total protein concentration. I t should be noted that the linear portion of the AE vs. -log [SH] plot approaches the Nernstian response for a 1:l interaction of sulfhydryl with Ag+; e.g., the electrode potential is being determined by the sulfhydryl content of the protein sample. Earlier titration experiments (11) on reduced ribonuclease A had also shown good agreement with these findings.
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
(1) (2) (3) (4)
R. P. Buck, Anal. Chem., 48, 23R (1976). G. J. Moody and J. D. R. Thomas, Analyst (London), 100, 609 (1975). S-t Chiu and L. Paszner, Anal. Chem., 47, 1910 (1975). M. T. Neshkova, V. P. Izvekov, M. K. Papay, K. Toth, and E. Pungor, Anal. Cbim. Acta, 75, 439 (1975). (5) P. K. C. Tseng and W. F. Gutknecht, Anal. Chem., 47, 2316 (1975). (6) L. C. Gruen, Biochim. Biophys. Acta, 386, 270 (1975). (7) B. S. Harrap and L. C. Gruen, Anal. Biochem., 42, 377 (1971). (8) W. Selig, Mikrochim. Acta, 73, 453 (1973). (9) P. W. Alexander and G. A. Rechnitz, Anal. Chem., 46, 250 (1974). (IO) P. W. Alexander and G. A. Rechnitz, Anal. Chem., 46, 860 (1974). (11) B. S. Harrap and L. C. Gruen, Anal. Biocbem., 42, 398 (1971). (12) "Sulfhydryl and Disulfide Groups of Proteins", Y. M. Torchinskii and H. B. F. Dixon, Consultants Bureau, New York, 1974. (13) H. Burton, Biocbim. Biophys. Acta, 29, 193 (1958). (14) L. 0. Anderson, J. Polym. Sci., Part A-1, I O , 1963 (1972). (15) I. M. Kolthoff and W. Stricks, J. Am. Cbem. SOC.,72, 1952 (1950). (16) L. A. E. Sluyterman, Biochim. Biopbys. Acta, 25, 402 (1957). (17) L. A. E. Sluyterman, Anal. Biochem., 14, 317 (1966). (18) "Biochemist's Handbook", C. Long, Van Nostrand-Reinhold, New York, 1961. (19) G. L. Ellman, Arch. Biochem. Biophys., 82, 70 (1959). (20) C. 8. Anfinsen and E. Haber, J. Bid. Chem., 236, 1361 (1961). (21) F. H. White, Jr., J. Bid. Chem., 236, 1353 (1961). (22) "Selective Ion Sensitive Electrodes", G. J. Moody and J. D. R. Thomas, Merrow, Watford Herts, England, 1971. (23) W.E. Morf, G. Kahr, and W. Simon, Anal. Chem., 46, 1538 (1974). (24) D. J. Crombie, G. J. Moody, and J. D. R. Thomas, Anal. Cbim. Acta, 80, 1 (1975). (25) R. Cecil in "The Proteins", Vol. 1, H. Neurath, Ed., Academic Press, New York, 1965. (26) "Biochemistry", 2d ed., A. L. Lehninger, Worth, New York, 1975. (27) J. Carter, J. Bid. Chem., 234, 1705 (1959). (28) W.W. Cleland, Biochemistry, 3, 480 (1964). (29) D. S. Papastathopoulos and G. A. Rechnitz, Anal. Chim. Acta, 79, 19 (1975). (30) C. B. Anfinsen, E. Haber, M. Sela, and F. H. White, Jr., Proc. Natl. Acad. Sci., 47, 1309 (1961).
RECEIVEDfor review August 4, 1976. Accepted September 17, 1976. We gratefully acknowledge the support of a grant from the National Science Foundation.
