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Direct spectrophotometric estimation of ferrocyanide and its possible uses in sulfhydril oxidation studies
ANALYTICAL
Direct and
BIOCHEMISTRY
28, 230-242
(1969)
Spectrophotometric Its Possible
Estimation
Uses
in Sulfhydril
of
Ferrocyanide
Oxidation
Studies
D. K. KIDBY 3 Sub-department
of
Chemical Microbiology, Department University of Cambtidgye, England July
of
Biochemistry,
12, 1968
Methods for sulfhydril group measurement have been briefly reviewed by Leggett Bailey (1). The general field of techniques which rely upon the stoichiometry of the oxidation reaction: 2-SH
+ --s-S-
has been reviewed by Guzman Barron (2) and Chinard and Hellerman (3). The use of ferricyanide (hexacyanatoferrate(I1.I) ) as the oxidizing agent for this reaction has been discussed in some detail by Guzman Barron (2). The methods described (2) depend upon the estimation of the ferrocyanide (hexacyanatoferrate (II) ) formed, following oxidation of the sample by excess ferricyanide, by formation of Prussian blue (MTFeFe(CN) G, where MI is Na, K, or Rb, but not Li or Cs), which is then assayed spectrophotometrically. It is implicit that the method of estimating ferrocyanide does not itself lead to further oxidation of -S-Sbonds or ferrocyanide or to oxidation of groups other than -SH. Since the method depends upon the reaction of ferrocynaide with iron (III) ions under highly acid conditions, and the prior removal of the test substance if this is likely to precipitate under these conditions, it is doubtful that all of these criteria for the accuracy of the method are fulfilled. Furthermore, the noncorrespondence oxidation with those of the conditions required for the -SH required for the estimation of ferrocyanide limits the method to static measurements only. The present study describes the direct spectrophotometric measurement of ferrocyanide in the presence of excess ferricyanide under conditions similar to those required for stoichiometric re1 Present address : Department Guelph, Ontario, Canada.
of Microbiology, 230
University
of
Guelph,
DIRECT
ESTIMATION
OF
FERROCYANIDE
231
action of biological -SH groups with ferricyanide. The method is compared with the Prussian blue method (2) for the oxidation of the -SH group of cysteine. The work forms part of a preliminary study on -SH groups, by oxidative and other techniques, in yeast cell-wall material. The previously published ultraviolet spectra of ferrocyanide and ferricyanide (4-6) are not in good agreement, e.g., the molar extinction coefficients at 240 rnp, which is within the region of interest in the present study, differ by up to 70%. For this reason the ultraviolet spectra of ferrocyanide and ferricyanide were further examined prior to their use for measurement of ferrocyanide. METHODS
Spectra. A Cary 15 split-beam recording spectrophotometer was used for drawing spectra over the range 200 to 350 mp. For determination of molar extinction coefficients in the range 230 to 250 rnp, a Unicam SP 500 spectrophotometer was used. In all cases cells containing the appropriate solvent were used in the reference position, except for the drawing of difference spectra in which the corresponding ferricyanide solution was used. Temperature equilibrations were not usually performed, as equilibrated cell temperatures were found to be only lo to 2’ higher than the laboratory temperature of 20’ to 21’. For measurement of spectral shifts over the pH range 2 to 12, the single buffer of Theorell and Stenhagen (7) was selected, as this provided a minimum variation in ionized and unionized species. Solutions were freshly prepared and kept in the dark as much as possible. pH measurements. A glass electrode with automated temperature correction was calibrated for 20° at pH 4, ‘7, and 9. No correction was applied for the sodium-induced error (8) at highly alkaline pH. A 0.010 N NaOH solution yielded an uncorrected pH of 11.8. Kinetics of ferrocyanide oxidation. Ferrocyanide solutions were scanned continuously for periods of from 20 to 30 minutes at 22.5’ at 240, 304, and in some cases 227 m+ Rates were calculated using the measured extinction coefficients and it was assumed that the only reaction product was ferricyanide. The method is imprecise but was selected since it corresponded to the conditions under which the method was to be applied. Estimation of ferrocyanide and cysteine by the Prussian blue method. The method was as described by Guzman Barron (2) using the ferric sulfate reagent of Folin and Malmros (9). Its
232
D.
K.
KIDBY
use for estimating ferrocyanide in the ferricyanide reagent was as described by Anson (10). Estimation of ferrocyanide and cysteine by the direct method. For ferrocyanide estimation, the optical density of the sample was measured at 237 rnp and ferrocyanide calculated by reference to a calibration line obtained from measurements on solutions of the sample containing known quantities of ferrocyanide. For cysteine estimation the measurements were performed in the same way except that cysteine was used for preparation of the calibration line. Relative stability of methods for cysteine estimation. Reaction mixtures were assayed, returned to the unstoppered test tubes, and stored in the dark for 24 hours at room temperature prior to reassay. REAGENTS
Analytical-grade or repurified reagents and deionized water were used throughout. Ferricyanide. Potassium ferricyanide (Hopkin and Williams Ltd.) was dissolved in hot water and, following recrystallization, washed with cold water and ether, then dried under vacuum over P,O, in a light-tight container. A 0.534 M stock solution in water was prepared gravimetrically and stored in an air-tight bottle at 4O. Ferrocyanide content of this solution some weeks after its preparation, and near the completion of these experiments, was 0.018% of the ferricyanide present, on a molar basis. Ferrocyanide. Potassium ferrocyanide trihydrate (British Drug Houses) of stated 99% purity was not further purified. Solutions for spectral studies were made immediately prior to use but it was observed that a neutral solution in sodium phosphate buffer, stored in a stoppered bottle in the dark, showed no readily measurable decrease in ferrocyanide concentration following 20 hours at room temperature. Gum ghatti. A 200 gm sample of the crude preparation, containing plant debris (Hopkin and Williams), was largely dissolved, with heating, in 1000 ml of water, and squeezed through 2, then 4, and finally 8 layers, of muslin to remove most of the plant debris. The filtrate, heated to 50” to reduce the viscosity, was tipped slowly into 5 volumes of 96% ethanol precooled to 4”. The precipitated gum was collected on a stirring rod and washed with 96% ethanol and then ether, and dried under vacuum over P,O,.
DIRECT
ESTIMATION
OF
FERROCYANIDE
233
This preparation conformed with the purity criterion for its use as a clinical laboratory reagent (11). Cysteine . HCZ . H,O. Cysteine 1 HCl (Hopkin and Williams) was recrystallized according to du Vigneaud et CLI. (12)) washed with ice-cold 18% (v/v) HCl on a sintered-glass filter, and dried over P,O, and NaOH under vacuum in a light-tight container. Solutions were prepared immediately before use. Ez~fjcer s&ctions. A stock solution of pH 7.0, 0.40 M sodium phosphate was prepared from: Na,HPO, (British Drug Houses) / NaH,PO, .2H,O (Hopkin and Williams). The buffer of Theorell and Stenhagen (7) was prepared from: citric acid/@@ H,PO, (British Drug Houses)/NaOH, H,BO,, 35.4% HCl (Hopkin and Williams). Stock quantities at the required pH were prepared and finally diluted lo-fold, i.e., measurements were performed in solutions containing, in meq./lOOO ml: citric acid, 2.0; H3P0,, 2.0; H,BO,, 2.0; NaOH, 6.9; HCl, according to pH. Salt, acid, and alkali solutions. Stock solutions of 0.10 M reagents were prepared from: NaOH, 35.4% HCI, NH,Cl, CaCl, .6H?O, Na,SO, (Hopkin and Williams) ; MgCl, .6H,O, MnCl! .4H,O (British Drug Houses). Except for kinetic studies the ferrocyanide or ferricyanide was added immediately prior to measurement. RESULTS
The spectra of ferrocyanide and ferricyanide in water, 0.01 M sodium phosphate buffer at pH 7.0, 0.01 N HCl, and 0.01 N NaOH (Fig. 1) indicate relative stability at neutral or alkaline pH above 230 rnp. The wavelengths and extinction coefficients for maxima in the ferrocyanide spectra obtained in these solutions are given in Table 1. Positions of maxima in the difference spectra (not illustrated) for ferrocyanide and ferricyanide in these solutions are also recorded in Table 1. An examination of spectra over the pH range 2 to 12 (Fig. 2) reveals that little variation occurs in either the ferrocyanide OY the ferricyanide spectra above 230 rnp between pH 6 and 12. Solutions in pH 7.0 phosphate buffer were selected for further investigation. The ratios of extinction coefficients indicated that, for the estimation of ferrocyanide in the presence of excess ferricyanide, the region of interest lay between 230 and 250 rnp. On the basis of the ratios determined in this range (Table 2)) a wavelength
234
D. K. KIDRY
200
300
350 200
250
300
350
FIG. 1. Some comparisons between ferrocyanide (II) and ferricyanide (III) spectra at neutral, acid, and alkaline pH. (a) Solvent: Water. Potassium ferrocyanide and ferricyanide concentrations were 2.81 X 10-5 and 2.67 X 10-5 M, respectively. Optical densities were measured in 1.0 cm cells. (b) Solvent: 0.01 M sodium phosphate at pH 7.0. Remainder of protocol as for (a). (c) Solvent: 0.01 N hydrochloric acid. Remainder of protocol as for (a). (d) Solvent: 0.01 N sodium hydroxide. Remainder of protocol as for (a).
of 237 rnp was selected as being potentially suitable for measurement of ferrocyanide. However, before proceeding further, the stability of ferrocyanide under the selected conditions was compared with stability at pH 2 and 12 (Table 3). The discrepancy
DIRECT
ESTIMATION
Some Spectral Characteristics Solvent
H,O NazHPOl NaHpPOr, 0.01 M, pH 7.0 HCl, 0.01 N NaOH, 0.01 N
TABLE 1 of Ferrocyanide
Ferrocyanide x,.x, mp
235
OF FERROCYANIDE
and Ferricyanide
Ferrocyanide molar extinction coefficient X 10-b
Solutions D Difference x,.x,
spectra, m/.l
218 218
2.21 2.23
221 222
213 220
1.76 2.08
218 222
n Measurements were made with 1.0 cm cells containing 2.81 X lo-” M and 2.67 X 10-S M solutions of ferrocyanide and ferricyanide, respectively. Differenee spectra were drawn with the ferricyanide in the reference cell position.
Molar
Extinction
TABLE 2 Coefficients for Ferrocyanide and Ferricyanide from 230 to 250 mp a Ferrocyanide molar extinction x 10-4
230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250
1.50 1.40 1.33 1.22 1.14 1.10 1.03 0.968 0.887 0.830 0.777 0.741 0.674 0.632 0.593 0.559 0.527 0.491 0.467 0.441 0.422
Ferrieyanide molar extinction x 10-a
1.73 1.52 1.34 1.18 1.06 0.955 0.879 0.816 0.767 0.723 0.712 0.702 0.710 0.714 0.732 0.750 0.772 0.798 0.828 0.855 0.876
Solutions Ratio of extinction coefficients
8.7 9.2 9.9 10.3 10.8 11.5 11.7 11.9 11.6 11.5 10.9 10.6 9 .5 8.9 8.1 7.5 6.8 6.2 5.6 5.2 4.9
a Measurements were made with 1.0 cm cells containing 6.15 X 1O-6 IM and 5.34 X lo-4 M solutions of ferrocyanide and ferricyanide respectively, in 0.01 IM, pH 7.0, sodium phosphate.
236
D. , 1 PH
7 ’
K.
I “‘I
KIDBY ,,I
”
’ (
” PH
’
, 7 ”
7
7
06
(a)
6
I
250
I
1200
250
300
350
FIG. 2. Ferrocyanide and ferricyanide spectra over pH range 2 to 12. (a) 2.72 X 10-S M, in Theorell and Stenhagen (7) buffer. Potassium ferrocyanide, The positions of the curves are indicated, for each pH value, by arrows. Optical densities were Inensured in 1.0 cm cells. (b) Potassium ferrocyanide, 2.78 X l!)-” M. Remainder of protocol as fer (a). (c) Potassium ferricyanide, 2.67 X l()-: AI. Remainder of protocol as for (a). (d) Potassium ferricyanide, 2.67 X 1 n- ;I .I/. Remainder of protocol as for (a) .
between the calculated rates of oxidation of ferrocyanide by measurement of increases in optical density at 304 mP, assumed to be due to ferricyanide formation, and decreases in optical density at 240 mhL, assumed to be due to ferrocyanide oxidation, may be
DIRECT
Stability
ESTIMATION
of Ferrocyanide
TABLE 3 in Acid, Alkaline,
2 2 7 7 7 12 12 12
and Neutral
Optical density change/hr x 102
PH
240 304 240 304 227 240 304 227
237
OF FERROCYANIDE
-4.3” +3.2b OC OC Oc OC Oc Oc
Solution 4 Per cent of total ferrocyanidr oxidized/hrcl
9 21 <0.38 <0.3e <0.3’ <0.8* <0.8’:
a Rates of change of optical density were measured at 22.5” with 1.0 cm cells containing 9.35 X 10-j n/r solutions of ferrocyanide in the buffer of Theorell and Stenhagen (7). * Changes in optical density were measured continuously for periods of between 20 and 30 minutes. All observed changes were linear with time. c Measurements were made over periods of up to 152 minutes ;in each case. No measurable changes occurred. d Calculations were based upon the measured extinction coefficients of lferrocyanide and ferricyanide in the same solvent. c These figures represent the rate at which oxidation should have been detectable by the method used.
attributable to the imprecision of the method used. The kinetics of ferrocyanide oxidation under extremely acid conditions were not further investigated as these conditions were considered unsuitable for ferrocyanide estimation. Solutions of ferrocyanide conformed closely to the Beer-Lambert law at 237 rnp. The estimation of -SH of cysteine was attempted using a 5-, lo-, and 15-fold excess of ferricyanide and fair agreement was obtained between theoretical and observed increases in optical density (Table 4). A comparison of the Prussian blue method and measurement of optical density at 237 rnp was performed for a range of cysteine concentrations using varying concentrations of ferricyanide (Fig. 3). While the direct method yielded a linear response at both levels of ferricyanide, the Prussian blue method was satisfactory only at the recommended (2) level of ferricyanide. This latter observation was not further investigated. The stability of ferrocyanide, under the assay conditions selected, is apparently far greater than that of Prussian blue
238
D. K. KIDBY
01234567
x105M
Cysteine
FIG. 3. Comparison between direct and Prussian blue methods for estimation of ferrocyanide formed by reduction of ferricyanide by cysteine: (0) estimation by direct method at initial ferricyanide concentration of 5.34 X 10-4 M; (0) estimation by direct method at initial ferricynaide concentration of 2.67 X lo-4 M; (1) estimation by Prussian blue method at initial ferricyanide concentration of 1.07 X 10-Z M; (0) estimation by Prussian blue method at initial ferricyanide concentration of 5.34 X lo-4 M. Optical densities were measured in 1.0 cm cells at 237 and 710 mp for direct and Prussian blue methods, respectively. In the direct method, reaction mixtures initially contained potassium ferricyanide and cysteine hydrochloride, as indicated; sodium phosphate added as pH 7.0 buffer, 0.01 M. In the Prussian blue method, reaction mixtures initially contained potassium ferricyanide and cysteine hydrochloride, as indicated; sodium phosphate added as the pH 7.0 buffer, 0.008 M; sulfuric acid, 0.2 IV; ferric sulfate reagent (9)) 1 ml stock reagent in a final volume of 10 ml.
(Fig. 4). Except for the effect of the Mn (II) ion, the extinction coefficients of ferrocyanide and ferricyanide at 237 rnp are little effected by the cations and anions tested (Table 5). The relative lack of effect of chloride, phosphate, citrate, borate, and sodium ions may be inferred by comparing the spectra in water with buffered neutral and alkaline solutions, and with unbuffered alkaline solutions (Fig. 1 and 2). DISCUSSION
The spectra obtained in this work appear to be in agreement with those published by Cohen and Plane (6), rather than those
bIRECT
ESTIMATION
TABLE 4 Optical Density Changes in Ferricyanide Solutions Following Ferrieyanide:cysteine ratio
Predicted density
15 10 5
239
OF FERROCYANIDE
optical changeb
Addition Observed density
0.373 0.373 0.373
of Cysteine 0 optical change
0.365 0.370 0.387
n Measurements were made at 237 rnp with 1.0 cm cells containing 4.2 X lo+ M cysteine and 6.4 X lo-*, 4.3 X 10-4, or 2.1 X 1OW M ferricyanide buffered (7) at pH 7.0. Optical densities were measured at 2 and 5 minutes after addition of cysteine. No differences were observed over this period. b Predictions were based upon the measured extinction coefficients of ferrocyanide and ferricyanide, and the assumption of 1OO70 pure cysteine.HCl.HzO.
of Kortiim (4)) and Ibers and Davidson (5). In view of the relatively small spectral shifts produced by the addition of a variety of ions or variation in pH over the mildly acid to alkaline range, the reasons for the discrepancies between previously published spectra are not apparent. The effect of the Mn (II) ion is probably exceptional but perhaps not surprising since this is isoelectronic with the Fe (III) ion which, in acid solution, produces TABLE 5 Effects of Various Salts on Extinction Coefficients of Ferrocyanide and Ferricyanide at 237 rnp a Molar Salt
NH&l NH&l CaCb CaCb MgClz MgClz MnClr MnCb NazS04 Na2S04 Nil
extinction
coefficients
conc~tration,
Salt
Ferrocyanide x 10-s
0.100 0.010 0.100 0.010 0.100 0.010 0.100 0.010 0.100 0.010
9.19 9.20 9.10 9.12 8.85 8.96 7.92b 8.76b 9.13 9.12 9.03
0.822 0.828 0.835 0.842 0.842 0.842 1.00 0.918 0.833 0.838 0.836
a Measurements were made with 1.0 cm cells containing 5.92 X lo+ M and 5.34 X 10-h M solutions of ferrocyanide and ferricyanide, respectively. b Addition of ferrocyanide to MnCb solutions produced an immediate, and extremely fine, white precipitate which readily formed a visually homogeneous suspension.
240
D. K. KIDBY
0 0
1 1
1 2
! 3
1 4
5
6
1 7
x10m5M Cysteme FIG. 4. Comparison of stability of reaction mixtures between direct and Prussian blue methods: (0) estimation by direct method at initial ferricyanide concentration of 5.34 X 10-b M, with measurements made within 1 hour of reaction; (0) measurements of foregoing reaction mixtures after further 24 hours; ( n ) estimation by Prussian blue method at initial ferricyanide concentration of 1.07 X 10-Z M, with measurements made about 1 hour after reaction; (0) measurements of foregoing reaction mixtures after further 24 hours. Remainder of the protocol is as for Figure 3.
a very similar result, viz., a precipitate of Prussian blue. However, this is conjecture and has not been further examined. The additivity of light absorption for mixtures of ferrocyanide and ferricyanide was not directly studied but may be inferred from the linear increase in optical density in the presence of cysteine. Additivity of light absorption has been previously observed (5) for a 1: 1 mixture of ferrocyanide and ferricyanide but for wavelengths outside the region used in the present work. There have also been previous studies (6) on spectral shifts produced by association of various cations with ferrocyanide but the shifts were fairly small and were not studied below 250 rnp, Conformity to the Beer-Lambert law has also been examined at other wavelengths and, while no deviations are reported from two sources (4, 5)) another (6) reports a slight deviation for ferrocyanide but none for ferricyanide over the range 250 to 320 mp.
DIRECT
ESTIMATION
OF
FERROCYANIDG
241
The difference spectra for ferrocyanide and ferricyanide indicate the possibility of measurement of ferrocyanide in the region of 222 rnp but, since the ratio of extinction coefficients is rather low at this point, this would not be useful when a considerable excess of ferricyanide was present. In the latter case, the most useful region is between 235 and 239 mp. The direct method does possess certain disadvantages but in most instances these are readily overcome. It cannot be employed on solutions containing large excesses of substances which absorb strongly in the ultraviolet, e.g., its use in the presence of 7.5% trichloracetic acid is not practicable. The method is only slightly less sensitive than the Prussian blue method. The method appears to offer several advantages over the Prussian blue method. It is rapid, simple, and inexpensive. The stability is such that the reaction mixtures need not be measured for periods of some hours but this would require verification for the particular sample if this is to remain exposed to the oxidizing activity of ferricyanide. By the nature of the method and the observation of the instability of ferrocyanide in very acid solution, a higher precision and accuracy is inherent in the direct method. A potentially useful feature is that, while a large excess of the reagent can be tolerated, this is not necessary. Except insofar as they may effect the rate of reduction of ferricyanide, a number of commonly encountered ions should not effect the estimation of ferrocyanide. It is possible to assay small volumes at low sample concentrations since the ferricyanide reagent may be added as a concentrated solution in subdrop size quantities. One of the most important advantages that the method offers is that of performing direct kinetic measurements over a wide range of conditions including pH. The method is of sufficient sensitivity to permit the direct measurement of -SH oxidation in many proteins since the quantity of protein required for assay would not absorb more light than that which can be easily offset by use of a reference cell containing the sample. It is in the direct measurement of oxidation rates of sulfhydril groups that the method is likely to have its most useful application. The estimation of the number of sulfhydril groups in a particular protein is often more reliably achieved by other methods (1). The method, as described, has been used for the measurement of 2-mercaptoethanol and dithiothreitol (2, 3-dihydroxy-1, 4dithiolbutane) .
242
D. K. KIDBY SUMMARY
1. The direct spectrophotometry of ferrocyanide in the presence of ferricyanide has been examined. 2. Ferrocyanide at a concentration of 1 X lo-” M is easily measurable in the presence of a 50-fold excess of ferricaynide. 3. The measurement may be performed over a wide range of conditions, including those suited to the oxidation of most biological -SH groups 4. The method has been compared with the Prussian blue method for the estimation of oxidation of the -SH group of cysteine. 5. The method has many advantages over the Prussian blue method, including its possible use in direct kinetic measurements. ACKNOWLEDGMENTS I am indebted to the Commonwealth Scientific and Industrial Research Organization of Australia for a Post-doctoral Studentship, and to Professor E. F. Gale for support and research facilities. REFERENCES 1. LECETT BAILEY, J., “Techniques in Protein Chemistry,” 2nd ed. Elsevier, New York and London, 1967. 2. GUZMAN BARRON, E. S., Advan. Enzymol. 11,201 (1951). 3. CHINARD, F. D., AND HELLERMAN, L., i?~ “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. 1, p. 1. Interscience, New York and London, 1954. 4. KORT~M, G., 2. Physiol. Chem. 33,243 (1936). 5. IBERS, J. A., AND DAVIDSON, N., J. Am. Chem. Sot. 73,476 (1951). 6. COHEN, S. R., AND PLANE, A. R., J. Phys. Chemr 61,1096 (1957). 7. THEORELL, T., AND STENHAGEN, E., Biochem. 2.299,416 (1939). 8. VOGEL, A. I., “Quantitative Inorganic Analysis,” 3rd ed., p. 922. Longmans, London, 1962. 9. FOLIN, O., AND MALMROS, H. J., J. Biol. Chem. 83, 115 (1929). 10. ANSON, M. L., J. Gen., Physiol. 24,399 (1940). 11. “The Merck Index of Chemicals and Drugs” (P. G. Stecher, ed.), 7th ed. Merck & Co., Rahway, N. J., 1960. 12. DU VIGNEAUD, V., AUDRIETH, L. F., AND LORING, H. S., J. Am. Chem. Sot. 52.4500 (d30).
Direct and
BIOCHEMISTRY
28, 230-242
(1969)
Spectrophotometric Its Possible
Estimation
Uses
in Sulfhydril
of
Ferrocyanide
Oxidation
Studies
D. K. KIDBY 3 Sub-department
of
Chemical Microbiology, Department University of Cambtidgye, England July
of
Biochemistry,
12, 1968
Methods for sulfhydril group measurement have been briefly reviewed by Leggett Bailey (1). The general field of techniques which rely upon the stoichiometry of the oxidation reaction: 2-SH
+ --s-S-
has been reviewed by Guzman Barron (2) and Chinard and Hellerman (3). The use of ferricyanide (hexacyanatoferrate(I1.I) ) as the oxidizing agent for this reaction has been discussed in some detail by Guzman Barron (2). The methods described (2) depend upon the estimation of the ferrocyanide (hexacyanatoferrate (II) ) formed, following oxidation of the sample by excess ferricyanide, by formation of Prussian blue (MTFeFe(CN) G, where MI is Na, K, or Rb, but not Li or Cs), which is then assayed spectrophotometrically. It is implicit that the method of estimating ferrocyanide does not itself lead to further oxidation of -S-Sbonds or ferrocyanide or to oxidation of groups other than -SH. Since the method depends upon the reaction of ferrocynaide with iron (III) ions under highly acid conditions, and the prior removal of the test substance if this is likely to precipitate under these conditions, it is doubtful that all of these criteria for the accuracy of the method are fulfilled. Furthermore, the noncorrespondence oxidation with those of the conditions required for the -SH required for the estimation of ferrocyanide limits the method to static measurements only. The present study describes the direct spectrophotometric measurement of ferrocyanide in the presence of excess ferricyanide under conditions similar to those required for stoichiometric re1 Present address : Department Guelph, Ontario, Canada.
of Microbiology, 230
University
of
Guelph,
DIRECT
ESTIMATION
OF
FERROCYANIDE
231
action of biological -SH groups with ferricyanide. The method is compared with the Prussian blue method (2) for the oxidation of the -SH group of cysteine. The work forms part of a preliminary study on -SH groups, by oxidative and other techniques, in yeast cell-wall material. The previously published ultraviolet spectra of ferrocyanide and ferricyanide (4-6) are not in good agreement, e.g., the molar extinction coefficients at 240 rnp, which is within the region of interest in the present study, differ by up to 70%. For this reason the ultraviolet spectra of ferrocyanide and ferricyanide were further examined prior to their use for measurement of ferrocyanide. METHODS
Spectra. A Cary 15 split-beam recording spectrophotometer was used for drawing spectra over the range 200 to 350 mp. For determination of molar extinction coefficients in the range 230 to 250 rnp, a Unicam SP 500 spectrophotometer was used. In all cases cells containing the appropriate solvent were used in the reference position, except for the drawing of difference spectra in which the corresponding ferricyanide solution was used. Temperature equilibrations were not usually performed, as equilibrated cell temperatures were found to be only lo to 2’ higher than the laboratory temperature of 20’ to 21’. For measurement of spectral shifts over the pH range 2 to 12, the single buffer of Theorell and Stenhagen (7) was selected, as this provided a minimum variation in ionized and unionized species. Solutions were freshly prepared and kept in the dark as much as possible. pH measurements. A glass electrode with automated temperature correction was calibrated for 20° at pH 4, ‘7, and 9. No correction was applied for the sodium-induced error (8) at highly alkaline pH. A 0.010 N NaOH solution yielded an uncorrected pH of 11.8. Kinetics of ferrocyanide oxidation. Ferrocyanide solutions were scanned continuously for periods of from 20 to 30 minutes at 22.5’ at 240, 304, and in some cases 227 m+ Rates were calculated using the measured extinction coefficients and it was assumed that the only reaction product was ferricyanide. The method is imprecise but was selected since it corresponded to the conditions under which the method was to be applied. Estimation of ferrocyanide and cysteine by the Prussian blue method. The method was as described by Guzman Barron (2) using the ferric sulfate reagent of Folin and Malmros (9). Its
232
D.
K.
KIDBY
use for estimating ferrocyanide in the ferricyanide reagent was as described by Anson (10). Estimation of ferrocyanide and cysteine by the direct method. For ferrocyanide estimation, the optical density of the sample was measured at 237 rnp and ferrocyanide calculated by reference to a calibration line obtained from measurements on solutions of the sample containing known quantities of ferrocyanide. For cysteine estimation the measurements were performed in the same way except that cysteine was used for preparation of the calibration line. Relative stability of methods for cysteine estimation. Reaction mixtures were assayed, returned to the unstoppered test tubes, and stored in the dark for 24 hours at room temperature prior to reassay. REAGENTS
Analytical-grade or repurified reagents and deionized water were used throughout. Ferricyanide. Potassium ferricyanide (Hopkin and Williams Ltd.) was dissolved in hot water and, following recrystallization, washed with cold water and ether, then dried under vacuum over P,O, in a light-tight container. A 0.534 M stock solution in water was prepared gravimetrically and stored in an air-tight bottle at 4O. Ferrocyanide content of this solution some weeks after its preparation, and near the completion of these experiments, was 0.018% of the ferricyanide present, on a molar basis. Ferrocyanide. Potassium ferrocyanide trihydrate (British Drug Houses) of stated 99% purity was not further purified. Solutions for spectral studies were made immediately prior to use but it was observed that a neutral solution in sodium phosphate buffer, stored in a stoppered bottle in the dark, showed no readily measurable decrease in ferrocyanide concentration following 20 hours at room temperature. Gum ghatti. A 200 gm sample of the crude preparation, containing plant debris (Hopkin and Williams), was largely dissolved, with heating, in 1000 ml of water, and squeezed through 2, then 4, and finally 8 layers, of muslin to remove most of the plant debris. The filtrate, heated to 50” to reduce the viscosity, was tipped slowly into 5 volumes of 96% ethanol precooled to 4”. The precipitated gum was collected on a stirring rod and washed with 96% ethanol and then ether, and dried under vacuum over P,O,.
DIRECT
ESTIMATION
OF
FERROCYANIDE
233
This preparation conformed with the purity criterion for its use as a clinical laboratory reagent (11). Cysteine . HCZ . H,O. Cysteine 1 HCl (Hopkin and Williams) was recrystallized according to du Vigneaud et CLI. (12)) washed with ice-cold 18% (v/v) HCl on a sintered-glass filter, and dried over P,O, and NaOH under vacuum in a light-tight container. Solutions were prepared immediately before use. Ez~fjcer s&ctions. A stock solution of pH 7.0, 0.40 M sodium phosphate was prepared from: Na,HPO, (British Drug Houses) / NaH,PO, .2H,O (Hopkin and Williams). The buffer of Theorell and Stenhagen (7) was prepared from: citric acid/@@ H,PO, (British Drug Houses)/NaOH, H,BO,, 35.4% HCl (Hopkin and Williams). Stock quantities at the required pH were prepared and finally diluted lo-fold, i.e., measurements were performed in solutions containing, in meq./lOOO ml: citric acid, 2.0; H3P0,, 2.0; H,BO,, 2.0; NaOH, 6.9; HCl, according to pH. Salt, acid, and alkali solutions. Stock solutions of 0.10 M reagents were prepared from: NaOH, 35.4% HCI, NH,Cl, CaCl, .6H?O, Na,SO, (Hopkin and Williams) ; MgCl, .6H,O, MnCl! .4H,O (British Drug Houses). Except for kinetic studies the ferrocyanide or ferricyanide was added immediately prior to measurement. RESULTS
The spectra of ferrocyanide and ferricyanide in water, 0.01 M sodium phosphate buffer at pH 7.0, 0.01 N HCl, and 0.01 N NaOH (Fig. 1) indicate relative stability at neutral or alkaline pH above 230 rnp. The wavelengths and extinction coefficients for maxima in the ferrocyanide spectra obtained in these solutions are given in Table 1. Positions of maxima in the difference spectra (not illustrated) for ferrocyanide and ferricyanide in these solutions are also recorded in Table 1. An examination of spectra over the pH range 2 to 12 (Fig. 2) reveals that little variation occurs in either the ferrocyanide OY the ferricyanide spectra above 230 rnp between pH 6 and 12. Solutions in pH 7.0 phosphate buffer were selected for further investigation. The ratios of extinction coefficients indicated that, for the estimation of ferrocyanide in the presence of excess ferricyanide, the region of interest lay between 230 and 250 rnp. On the basis of the ratios determined in this range (Table 2)) a wavelength
234
D. K. KIDRY
200
300
350 200
250
300
350
FIG. 1. Some comparisons between ferrocyanide (II) and ferricyanide (III) spectra at neutral, acid, and alkaline pH. (a) Solvent: Water. Potassium ferrocyanide and ferricyanide concentrations were 2.81 X 10-5 and 2.67 X 10-5 M, respectively. Optical densities were measured in 1.0 cm cells. (b) Solvent: 0.01 M sodium phosphate at pH 7.0. Remainder of protocol as for (a). (c) Solvent: 0.01 N hydrochloric acid. Remainder of protocol as for (a). (d) Solvent: 0.01 N sodium hydroxide. Remainder of protocol as for (a).
of 237 rnp was selected as being potentially suitable for measurement of ferrocyanide. However, before proceeding further, the stability of ferrocyanide under the selected conditions was compared with stability at pH 2 and 12 (Table 3). The discrepancy
DIRECT
ESTIMATION
Some Spectral Characteristics Solvent
H,O NazHPOl NaHpPOr, 0.01 M, pH 7.0 HCl, 0.01 N NaOH, 0.01 N
TABLE 1 of Ferrocyanide
Ferrocyanide x,.x, mp
235
OF FERROCYANIDE
and Ferricyanide
Ferrocyanide molar extinction coefficient X 10-b
Solutions D Difference x,.x,
spectra, m/.l
218 218
2.21 2.23
221 222
213 220
1.76 2.08
218 222
n Measurements were made with 1.0 cm cells containing 2.81 X lo-” M and 2.67 X 10-S M solutions of ferrocyanide and ferricyanide, respectively. Differenee spectra were drawn with the ferricyanide in the reference cell position.
Molar
Extinction
TABLE 2 Coefficients for Ferrocyanide and Ferricyanide from 230 to 250 mp a Ferrocyanide molar extinction x 10-4
230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250
1.50 1.40 1.33 1.22 1.14 1.10 1.03 0.968 0.887 0.830 0.777 0.741 0.674 0.632 0.593 0.559 0.527 0.491 0.467 0.441 0.422
Ferrieyanide molar extinction x 10-a
1.73 1.52 1.34 1.18 1.06 0.955 0.879 0.816 0.767 0.723 0.712 0.702 0.710 0.714 0.732 0.750 0.772 0.798 0.828 0.855 0.876
Solutions Ratio of extinction coefficients
8.7 9.2 9.9 10.3 10.8 11.5 11.7 11.9 11.6 11.5 10.9 10.6 9 .5 8.9 8.1 7.5 6.8 6.2 5.6 5.2 4.9
a Measurements were made with 1.0 cm cells containing 6.15 X 1O-6 IM and 5.34 X lo-4 M solutions of ferrocyanide and ferricyanide respectively, in 0.01 IM, pH 7.0, sodium phosphate.
236
D. , 1 PH
7 ’
K.
I “‘I
KIDBY ,,I
”
’ (
” PH
’
, 7 ”
7
7
06
(a)
6
I
250
I
1200
250
300
350
FIG. 2. Ferrocyanide and ferricyanide spectra over pH range 2 to 12. (a) 2.72 X 10-S M, in Theorell and Stenhagen (7) buffer. Potassium ferrocyanide, The positions of the curves are indicated, for each pH value, by arrows. Optical densities were Inensured in 1.0 cm cells. (b) Potassium ferrocyanide, 2.78 X l!)-” M. Remainder of protocol as fer (a). (c) Potassium ferricyanide, 2.67 X l()-: AI. Remainder of protocol as for (a). (d) Potassium ferricyanide, 2.67 X 1 n- ;I .I/. Remainder of protocol as for (a) .
between the calculated rates of oxidation of ferrocyanide by measurement of increases in optical density at 304 mP, assumed to be due to ferricyanide formation, and decreases in optical density at 240 mhL, assumed to be due to ferrocyanide oxidation, may be
DIRECT
Stability
ESTIMATION
of Ferrocyanide
TABLE 3 in Acid, Alkaline,
2 2 7 7 7 12 12 12
and Neutral
Optical density change/hr x 102
PH
240 304 240 304 227 240 304 227
237
OF FERROCYANIDE
-4.3” +3.2b OC OC Oc OC Oc Oc
Solution 4 Per cent of total ferrocyanidr oxidized/hrcl
9 21 <0.38 <0.3e <0.3’ <0.8* <0.8’:
a Rates of change of optical density were measured at 22.5” with 1.0 cm cells containing 9.35 X 10-j n/r solutions of ferrocyanide in the buffer of Theorell and Stenhagen (7). * Changes in optical density were measured continuously for periods of between 20 and 30 minutes. All observed changes were linear with time. c Measurements were made over periods of up to 152 minutes ;in each case. No measurable changes occurred. d Calculations were based upon the measured extinction coefficients of lferrocyanide and ferricyanide in the same solvent. c These figures represent the rate at which oxidation should have been detectable by the method used.
attributable to the imprecision of the method used. The kinetics of ferrocyanide oxidation under extremely acid conditions were not further investigated as these conditions were considered unsuitable for ferrocyanide estimation. Solutions of ferrocyanide conformed closely to the Beer-Lambert law at 237 rnp. The estimation of -SH of cysteine was attempted using a 5-, lo-, and 15-fold excess of ferricyanide and fair agreement was obtained between theoretical and observed increases in optical density (Table 4). A comparison of the Prussian blue method and measurement of optical density at 237 rnp was performed for a range of cysteine concentrations using varying concentrations of ferricyanide (Fig. 3). While the direct method yielded a linear response at both levels of ferricyanide, the Prussian blue method was satisfactory only at the recommended (2) level of ferricyanide. This latter observation was not further investigated. The stability of ferrocyanide, under the assay conditions selected, is apparently far greater than that of Prussian blue
238
D. K. KIDBY
01234567
x105M
Cysteine
FIG. 3. Comparison between direct and Prussian blue methods for estimation of ferrocyanide formed by reduction of ferricyanide by cysteine: (0) estimation by direct method at initial ferricyanide concentration of 5.34 X 10-4 M; (0) estimation by direct method at initial ferricynaide concentration of 2.67 X lo-4 M; (1) estimation by Prussian blue method at initial ferricyanide concentration of 1.07 X 10-Z M; (0) estimation by Prussian blue method at initial ferricyanide concentration of 5.34 X lo-4 M. Optical densities were measured in 1.0 cm cells at 237 and 710 mp for direct and Prussian blue methods, respectively. In the direct method, reaction mixtures initially contained potassium ferricyanide and cysteine hydrochloride, as indicated; sodium phosphate added as pH 7.0 buffer, 0.01 M. In the Prussian blue method, reaction mixtures initially contained potassium ferricyanide and cysteine hydrochloride, as indicated; sodium phosphate added as the pH 7.0 buffer, 0.008 M; sulfuric acid, 0.2 IV; ferric sulfate reagent (9)) 1 ml stock reagent in a final volume of 10 ml.
(Fig. 4). Except for the effect of the Mn (II) ion, the extinction coefficients of ferrocyanide and ferricyanide at 237 rnp are little effected by the cations and anions tested (Table 5). The relative lack of effect of chloride, phosphate, citrate, borate, and sodium ions may be inferred by comparing the spectra in water with buffered neutral and alkaline solutions, and with unbuffered alkaline solutions (Fig. 1 and 2). DISCUSSION
The spectra obtained in this work appear to be in agreement with those published by Cohen and Plane (6), rather than those
bIRECT
ESTIMATION
TABLE 4 Optical Density Changes in Ferricyanide Solutions Following Ferrieyanide:cysteine ratio
Predicted density
15 10 5
239
OF FERROCYANIDE
optical changeb
Addition Observed density
0.373 0.373 0.373
of Cysteine 0 optical change
0.365 0.370 0.387
n Measurements were made at 237 rnp with 1.0 cm cells containing 4.2 X lo+ M cysteine and 6.4 X lo-*, 4.3 X 10-4, or 2.1 X 1OW M ferricyanide buffered (7) at pH 7.0. Optical densities were measured at 2 and 5 minutes after addition of cysteine. No differences were observed over this period. b Predictions were based upon the measured extinction coefficients of ferrocyanide and ferricyanide, and the assumption of 1OO70 pure cysteine.HCl.HzO.
of Kortiim (4)) and Ibers and Davidson (5). In view of the relatively small spectral shifts produced by the addition of a variety of ions or variation in pH over the mildly acid to alkaline range, the reasons for the discrepancies between previously published spectra are not apparent. The effect of the Mn (II) ion is probably exceptional but perhaps not surprising since this is isoelectronic with the Fe (III) ion which, in acid solution, produces TABLE 5 Effects of Various Salts on Extinction Coefficients of Ferrocyanide and Ferricyanide at 237 rnp a Molar Salt
NH&l NH&l CaCb CaCb MgClz MgClz MnClr MnCb NazS04 Na2S04 Nil
extinction
coefficients
conc~tration,
Salt
Ferrocyanide x 10-s
0.100 0.010 0.100 0.010 0.100 0.010 0.100 0.010 0.100 0.010
9.19 9.20 9.10 9.12 8.85 8.96 7.92b 8.76b 9.13 9.12 9.03
0.822 0.828 0.835 0.842 0.842 0.842 1.00 0.918 0.833 0.838 0.836
a Measurements were made with 1.0 cm cells containing 5.92 X lo+ M and 5.34 X 10-h M solutions of ferrocyanide and ferricyanide, respectively. b Addition of ferrocyanide to MnCb solutions produced an immediate, and extremely fine, white precipitate which readily formed a visually homogeneous suspension.
240
D. K. KIDBY
0 0
1 1
1 2
! 3
1 4
5
6
1 7
x10m5M Cysteme FIG. 4. Comparison of stability of reaction mixtures between direct and Prussian blue methods: (0) estimation by direct method at initial ferricyanide concentration of 5.34 X 10-b M, with measurements made within 1 hour of reaction; (0) measurements of foregoing reaction mixtures after further 24 hours; ( n ) estimation by Prussian blue method at initial ferricyanide concentration of 1.07 X 10-Z M, with measurements made about 1 hour after reaction; (0) measurements of foregoing reaction mixtures after further 24 hours. Remainder of the protocol is as for Figure 3.
a very similar result, viz., a precipitate of Prussian blue. However, this is conjecture and has not been further examined. The additivity of light absorption for mixtures of ferrocyanide and ferricyanide was not directly studied but may be inferred from the linear increase in optical density in the presence of cysteine. Additivity of light absorption has been previously observed (5) for a 1: 1 mixture of ferrocyanide and ferricyanide but for wavelengths outside the region used in the present work. There have also been previous studies (6) on spectral shifts produced by association of various cations with ferrocyanide but the shifts were fairly small and were not studied below 250 rnp, Conformity to the Beer-Lambert law has also been examined at other wavelengths and, while no deviations are reported from two sources (4, 5)) another (6) reports a slight deviation for ferrocyanide but none for ferricyanide over the range 250 to 320 mp.
DIRECT
ESTIMATION
OF
FERROCYANIDG
241
The difference spectra for ferrocyanide and ferricyanide indicate the possibility of measurement of ferrocyanide in the region of 222 rnp but, since the ratio of extinction coefficients is rather low at this point, this would not be useful when a considerable excess of ferricyanide was present. In the latter case, the most useful region is between 235 and 239 mp. The direct method does possess certain disadvantages but in most instances these are readily overcome. It cannot be employed on solutions containing large excesses of substances which absorb strongly in the ultraviolet, e.g., its use in the presence of 7.5% trichloracetic acid is not practicable. The method is only slightly less sensitive than the Prussian blue method. The method appears to offer several advantages over the Prussian blue method. It is rapid, simple, and inexpensive. The stability is such that the reaction mixtures need not be measured for periods of some hours but this would require verification for the particular sample if this is to remain exposed to the oxidizing activity of ferricyanide. By the nature of the method and the observation of the instability of ferrocyanide in very acid solution, a higher precision and accuracy is inherent in the direct method. A potentially useful feature is that, while a large excess of the reagent can be tolerated, this is not necessary. Except insofar as they may effect the rate of reduction of ferricyanide, a number of commonly encountered ions should not effect the estimation of ferrocyanide. It is possible to assay small volumes at low sample concentrations since the ferricyanide reagent may be added as a concentrated solution in subdrop size quantities. One of the most important advantages that the method offers is that of performing direct kinetic measurements over a wide range of conditions including pH. The method is of sufficient sensitivity to permit the direct measurement of -SH oxidation in many proteins since the quantity of protein required for assay would not absorb more light than that which can be easily offset by use of a reference cell containing the sample. It is in the direct measurement of oxidation rates of sulfhydril groups that the method is likely to have its most useful application. The estimation of the number of sulfhydril groups in a particular protein is often more reliably achieved by other methods (1). The method, as described, has been used for the measurement of 2-mercaptoethanol and dithiothreitol (2, 3-dihydroxy-1, 4dithiolbutane) .
242
D. K. KIDBY SUMMARY
1. The direct spectrophotometry of ferrocyanide in the presence of ferricyanide has been examined. 2. Ferrocyanide at a concentration of 1 X lo-” M is easily measurable in the presence of a 50-fold excess of ferricaynide. 3. The measurement may be performed over a wide range of conditions, including those suited to the oxidation of most biological -SH groups 4. The method has been compared with the Prussian blue method for the estimation of oxidation of the -SH group of cysteine. 5. The method has many advantages over the Prussian blue method, including its possible use in direct kinetic measurements. ACKNOWLEDGMENTS I am indebted to the Commonwealth Scientific and Industrial Research Organization of Australia for a Post-doctoral Studentship, and to Professor E. F. Gale for support and research facilities. REFERENCES 1. LECETT BAILEY, J., “Techniques in Protein Chemistry,” 2nd ed. Elsevier, New York and London, 1967. 2. GUZMAN BARRON, E. S., Advan. Enzymol. 11,201 (1951). 3. CHINARD, F. D., AND HELLERMAN, L., i?~ “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. 1, p. 1. Interscience, New York and London, 1954. 4. KORT~M, G., 2. Physiol. Chem. 33,243 (1936). 5. IBERS, J. A., AND DAVIDSON, N., J. Am. Chem. Sot. 73,476 (1951). 6. COHEN, S. R., AND PLANE, A. R., J. Phys. Chemr 61,1096 (1957). 7. THEORELL, T., AND STENHAGEN, E., Biochem. 2.299,416 (1939). 8. VOGEL, A. I., “Quantitative Inorganic Analysis,” 3rd ed., p. 922. Longmans, London, 1962. 9. FOLIN, O., AND MALMROS, H. J., J. Biol. Chem. 83, 115 (1929). 10. ANSON, M. L., J. Gen., Physiol. 24,399 (1940). 11. “The Merck Index of Chemicals and Drugs” (P. G. Stecher, ed.), 7th ed. Merck & Co., Rahway, N. J., 1960. 12. DU VIGNEAUD, V., AUDRIETH, L. F., AND LORING, H. S., J. Am. Chem. Sot. 52.4500 (d30).