ANALYTICAL EDITION
April, 1945 LITERATURE CITED
Acree, F., Jr., J . Assoc. Oficial A&. Chem., 3, 648-51 (1941). Beet, A . E., and Belcher, R., Mikrochemie, 24, 145-8 (1938). Belcher, R., and Godbert, A. L., J . SOC.Chem. I d . , 60, 196-8 (1941).
Blatt, A. H., “Organic Syntheses”, Collective Vol. 11, p. 120, New York, John Wiley & Sons, 1944. Block, R. J., “Determination of Amino Acids”, Burgess Publishing Co., Minneapolis, Minn., 1942. Block, R. J., personal communication. Bradstreet, R. B., IND.ESG. CHEX..,ANAL.ED.,12, 657 (1940). Cerbelaud, R., and Bayard, C., “Manuel Clinique d’iinalyses Bacteriologiques”, p. 189, Paris, Bayard, 1907. Chibnall, A. C., Rees, hl. W., and Williams, E. F. Biochem. J., 37, 354-9 (1943).
Clark, E. P., IND.ENG.CHEM., .4NAL. ED., 10,677 (1938). Clark, E. P., J . Assoc. Oficial Agr. Chem., 24, 641-7 (1941). Cohn, E. J., and Hendry, J. L., J . Gen. Phyaiol., 5 , 521 (1923). Dakin, H. D., J . B i d . Chem., 44, 499-529 (1920). Dewey, B. T., and Witt, N. F., J . A m . Pharm. Assoc., 32, 65 (1943).
Drechsel, J . , J . prakt. Chem. ( 2 ) ,39, 425 (1889). Drummond, J. C., Biochem. J . , 12, 5-24 (1918). Dupray, h i . , J . Lab. Clin. &Wed.,12, 387 (1926). Folin, O., and Wu, H., J . Bid. Chem., 38, 81 (1919). Gortner, R. A . , and Hoffman, W. F., Ibid., 70,457 (1926). Hotchkiss, R. D., and Dubos, R., Ibid., 141, 155-62 (1941). Jonnard, R., and Fischer, L. O., Abstracts of Papers, AM. CHEM. SOC. Meeting, Pittsburgh, p . 21-B (Sept., 1943). LRUrO. M .F., IND.ENQ.C H E M . , .4NiL. ED.,3,401-2 (1931).
(23) (24)
249
Loeb, J., J . Gen. Phusiol., 3, 547 (192C-21). Xlears. B., and Hussey, R. E., IXD.ENG.CHEM.,A N A L .ED., 13, 1054 (1942).
( 2 6 ) Pepkovitz, L. P., Prince, A . L., and Bear, F. E., I b i d . , 14, 856-7 (1942).
(26)
Peters, J. P., and Van Slyke, D. D., “Quantitative Clinical Chemical Methods”, p . 519, Baltimore, Williams 8: Kilkins,
(Pi)
Ramsdall, G. A . , and Whittier, E. C., J . Bid. Chem., 151, 413-19
1932. ( 1944).
(28) Redemann, C. E., IND.Esq. CHEM.,Aix.4~. Eo., 11, 635-7 (1939). (29) (30)
St. John, J. L., J . Assoc. Oficial Agr. Chem., 24, 932-5 ~ 1 9 4 1 ~ . Scarrow, J. A., and Allen, C. F. H., Can. J . Research. 10, 73-6
(31)
Taylor, W. H., and Smith, G. F., ISD. ENQ.CHEX.,.\NL.
(1934).
En.,
14, 437-9 (1942). (32)
Van Slyke, D. D., Hiller, .1.,and Dillon, R . T., J . B d Chem.,
(33)
Villiers, A., and Moreau-Talon, -I,, Bull.
146, 137-57 (1942). SOC.
chirn., 23, 308
(1918). (34) (35) (36)
Wagner, E. C., IND. ENG.CHEM.,ANAL.ED.,12, i i l - 2 (1940). Warner, R., J . Am. C h m . SOC.,66, 1725-31 (1944). Wichmann, H. J., J . Assoc. Oficial Agr. Chem., 26, 522-59
(37)
Wicks: L. E., and Firminger, H. I., IND.ENG.CHEsr.. Ax.9~.
(1943).
ED.,14, 760-2
(1942).
PREBESTED in part before t h e Division of Biological Chemistry a t the 108tll Meeting of t h e AMERICAN CHE\IICAL SOCIETY, N e w York, N . 1.
Determination of Copper in Copper Proteins Using the Dropping M e r c u r y Electr0.de STANLEY R. AMES’
AND
CHARLES R. DAWSON N. Y.
Department of Chemistry, Columbia University, N e w York,
A specific method for the determination of copper in copper proteins involves use of the dropping mercury electrode after an acid extraction of the copper. The base solution for analysis is an acid sodium citrate buffer containing 0.005% fuchsin as a maximum suppressor. The copper can b e quantitatively extracted and the presence of native protein and protein breakdown products has been shown to b e permissible within prescribed limits. The method therefore elimi-
D
URIKG the past decade, interest in the role of minute amounts of copper in various biological processes has been revived as a result of the isolation of a number of copper proteins. These conjugated proteins, containing, in so far as is known at present, only copper as the nonprotein constituent, have been isolated from both plant and animal sources (3, 4,12, 16, f7,19, 21, 22, 29). As we gain knowledge of the importance of these copper proteins in plant and animal tissues, the need for a specific method for the rapid and precise microdetermination of copper in copper protein solutions becomes apparent. Several methods (2, 6 23 34) are available for the anaylsis of copper in plant and antma! tissue extracts, but most of these involve an ashing procedure which is time-consuming and productive of errors through contamination. For laboratories havin re$irometer equipment (6),the manometric method of d r b u r g (33) as modified b y Warburg and Krebs (34) has met with approval, since i t does not involve a long ashing procedure. This method has the advantage of speed, and has been extensively used in these laboratories (3,4, 16, f7,21, 2 2 ) . This experience has revealed, however, t h a t the precision of the method is not all that could be desired. T o obtain reliable results, the operator must be highly skilled in the technique. Even then a series of 1 Present address, Department of Madison, Wis.
Biochemistry, University of Wisconsin,
nates the necessity of a tedious arhing procedure. The half-wave potential at 25.0’ C. for cupric ion in the above base solution i s -0.18 volt vs. the saturated calomel electrode. The diffusion coefFicient at 25.0’ C. OF the cupric citrate complex in the above Practical limits of the medium is equal to 0.43 X 10-6 cm.2 sec.-l. method are from 1 to 75 to 100 micrograms per ml. of copper in the base solution with an average deviation of + 3%.
determinations on the same sample shows n mean deviation of about *1070. Recently, Reed and Cummings (23) have reported that copper in plant materials can be satisfactorily determined by use of the dropping mercury electrode. The earlier investigations of the rlarographic analysis for copper in the ash of hioloqical material y Kamegai (a), Mandai ( l a ) , Shoji (N),and Roncato and Bassani (24) were of a preliminary nature. Thanheiser and Maassen ( 3 2 ) , Such9 (31), and Stout, Levy, and,Williams (SO) have suggested procedures for the polarographic analysis of co er in steel, brass, and other nonbiological materials. prepare the sample for analysis according to the method of Reed and Cummings ( 6 3 ) , the lant material is subjected to a rather long and tedious wet-asfiing procedure, involving concentrated nitric, sulfuric, and perchloric acids. Interfering iron is then removed by precipitation with ammonium hydroxide. An acid sodium citrate buffer, containing acid fuchsin m a maximum suppressor, is used as the regulating solution for the polarographic analysis. The limits of the method, using a 1-grani sample of plant material, were reported as from 200 to 0 . 2 microgram of copper. T h e precision was not estimated and the temperature was not given.
E
It has been well established that copper proteins lose their copper in acid solutions-Le., below a p H of 3.0 (3,4,‘7, 1 2 ) . Copper proteins contain copper in both the cupric and cuprous form, but the copper in solution after an acid extraction would t.end to be in the cupric form, since cuprous copper is readily
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
250
oxidized in aqueous solution (13). I n the interest of speeding up the analysis and eliminatihg possible sources of contamination, i t seemed of value, therefore, to investigate the possibility. of substituting an acid extraction in place of the more tedious wetashing procedure in preparing purified and partially purified copper protein samples for analysis by means of the dropping mercury electrode. It is the purpose of this communication to report on an investigation of this nature. A satisfactory, rapid, and precise polarographic method for the microdetermination of copper in copper protein solutions using an acid extraction for the copper has been developed. Within certain limits of original protein concentration, the inftuence of extracted protein and protein breakdown products has been shown to introduce only a negligible error in the method. E X P E R I M E N T A L DETAILS
An Electropode, a manual olarograph produced by the Fbher Scientific Company, was u s e l The external arrangement of the cell and the electrodes was considerably modified, as diagramed in Figure 1. The cell containing the solution t o be analyzed was a U-shaped tube of 10-mm. glass tubin$, constricted to 1mm. a t the bend. It was about 6 cm. in height and contained a working volume of 0.5 to 1.0 ml. when an amount of mercury was added to give the maximum area of the quiet electrode. Into the em ty arm of the cell was inserted the lead for the quiet electrofe; and into the other arm, which contained the solution, were placed the dropping mercury capillary and tubes to bubble wet nitrogen through the solution.
Vol. 17, No. 4
blowing some of the sample partially out of the cell and introducin contaminating copper as’it drained back. %he cell and its contents were kept a t 25.0’ C. by means of water circulating from a constant-temperature bath. By raising or lowering the mercury reservoir, the drop rate was always adjusted to 3.5 seconds per drop when no potential was applied. The dropping electrode in all experiments was a section of tube drawn out to a capillary, whose internd diameter at the tip was about 0.03 mm. The r n * / a t ‘ / a value was obtained for each capillary. The bulbs, in which the copper protein solutions were evaporated to dryness, and in which the residues were then extracted with acid to remove the copper, were made in these laboratorins from 10-mm. Pyrex tubing (see Figure 2). They were of about 5-mi. capacity and weighed about 1.5 rams. To ensure thorough stirring during t%e extraction, these bulbs were slowly rotated by inserting the neck Figure 2 of the bulb into B piece of clean gum rubber tubing attached t o a variable-speed motor set at an angle of 60’ to the horiaontal. All volumes, except that of the fuchsin solution, were memured by meam of 1-ml. pipets graduated in hundredths, with the tips drawn out for slower delivery. The volume of fuchsin solution was determined by means of a 0.1-ml. pipet also graduated in hundredths. The effect of extraneous copper contamination was reduced to a minimum by cleanin4 all glassware in a routine manner. After soaking in chromic acid cleanin solution for several hours, the lassware was thoroughly cleanei by rinsing eight times in distifled water, four times in cold copper-free water, and four times in hot cop er-free water. [Distilled water was redistilled through a n all-$yrex apparatus having a 45cm. (18-inch) Vigreux type column that was electrically heated to about 140’ C. through a small region near the top of the column. This “hot zone” in the column prevented creeping of the condensation film, and water thus redistilled was found to be copper-free.] Each piece was protected from dust contamination by covering while draining and drying. The cleaning routine was carried out very carefully, since it was found that a minute contamination of cleaning solution rendered the analysis of no value. All solutions were made up in copper-free water. Five-tenths molar solutions of C.P. citric acid and C.P. sodium hydroxide were prepared without recrystallizing the solids, since any impurities present would be accounted for in the amplitude of the residual current correction. A 0.05% aqueous solution of fuchsin waa prepared as a maximum suppressor using the C.P. dye. The heptyl alcohol which served aa a frothing suppressor, wa8 obtained from the h a t m a n Kodak Company, and was found to be sufficiently pure in the amounts used, so that no further purification was necessary.
A N A L Y T I C A L PROCEDURE
Figure A. E.
I. Detail of Cell and Electrode Assembly
(-1
(+ anode lead anode Irad Nltroirn tuber E. Capill.ry C . Stopper
C.
H. Salutiaa - ..- ..L. Omid WKWY electrode M. Constant-tonperatwe water bath at 25.0’ I
C.
circulated with centrifnial P U ~ P
The nitrogen, used t o remove dissolved oxy en in the solution, was ordinary commercial tanked nitro en witg no attempt made to purify it. Experience has shown t t a t further purXcation is usually unnecessary (IO), but the nitrogen was bubbled slowly through water to saturate it with moisture, so prolonged bubbling would not change the concentration of the sample. Care was taken to bubble the nitrogen slowly, since there was danger of
The copper protein solution t o be analyzed was first thoroughly dialyzed for from 50 to 100 hours against repeated changes of copper-free water. A sample of the copper protein solution containing 1 to 100 micrograms of copper was placed in a small tared bulb (Figure 2) and evaporated to constant weight in an electric oven at 110’ C. The bulb was again weighed and the dry weight of protein in the sample thus determined. (The socalled dry weight of a protein sample and the true weight of protein present may not be the same. However, the term “dry weight” is in common usage.) The copper was extracted from the dried copper protein by adding a carefully measured amount (usually 0.50 ml.) of 0.5 M citric acid to the bulb containing the dried copper protein. The bulb was then rotated a t 60 to 120 r.p.m. as previously described for about 0.5 hour, or longer if a large residue (greater than 3 mg.) of protein was present. Then an equal volume of 0.5 M sodium hydroxide was accurately measured and added. The contents of the bulb were then mixed by rotation for 10 to 15 minutes, thus forming a sodium citrate buffer with a pH of sbout 4.0. If a large amount of precipitate of denatured protein appeared at this point, which was frequently the case when more than 3 mg. of protein were introduced into the bulb, the denatured protein was removed, since the particles interfered with drop formation in the subsequent analysis. This was readily accomplished by filtering an aliquot through a sintered-glass funnel, exercising due precautions not to introduce copper by contamination. A carefully measured aliquot (usually 0.50 ml.) of the solution was removed and placed in one arm of the polarographic cell. A carefully measured volume (usually 0.050 ml.) of a 0.05% aqueous solution of fuchsin, equal to approximately 10% of the
April, 1945
ANALYTICAL
Run 168. Copper Protein Preparation C211-228F2-Filtered 11. Dry-Weight Determination, 1.96-Ml.pample
+
V
I
I
11
Weight of protein bulb 5 2.55030 2.55028 Weight of bulb 5 2.54833 2.54833 Weight of protein 0.00197 0.00195 Sample contains 0.09Q6-m d r y wei h t per ml. Treatment of Samole. T%e 1.96-mf samnle was evanorated t o drvnaaa. 0.50 ml. of 0.6 M ciiric acid added, and th; bulb-rota~&-fo-r~36&'litas: Then, after adding 0.50 ml. of 0.5 M sodium hydroxide, rotation wan continued for 30 minutes. A 0.50-ml. aliquot wan removed and placed in the cell, and 0.050 ml. of 0.05% fuchsin wa8 added, bringing the volume t o 0.55 ml. Approximately 0.01 ml. of heptyl alcohol waa added and nitrogen bubbled for 20 minutes. 25.0" C. Capillary 10. Drop time, 3.5 seconda per drop with no potential applied. Analysis of Solution Sensitivity Applied Residual Shunt Ratio Potential Deflection Current Amplitude MicroMicroMicroampcrea amperes amperes x 102 x 102 Y o l l s o s . S.C.E. X 102
.
+O. 13
0-1 volt
1x
-
$0.03 -0.07 -0.17 -0.22 -0.27 -0.32 -0.37 -0.42 -0.47
-21.3 -12.2 6.1 9.3 22.2 24.2 25.5 26.3 27.2 27.6
+-
Setting
3.1 4.1 4.9 5.4 5.9
Average amplitude, 0.214 microampere 1 y Cu per ml. 0.0624 microampere (Table I). 0.214/0.0624 = 3.43 y/ml. Copper concentration Copper concentration in 0.55 ml. analysis volume Total copper in 0.55-ml. analysis volume Total copper in 0.50-ml. aliquot Total copper in 1.QO;ml. extraction volume Total copper of original sample Total dry weight of original sample 3.78 X 10-8 gram loo 0'196 % ' copper 1.95 X 10-1 gram
-
--
Figure 3.
=
0.0 microampere 21.1 21.4 21.4 21.8 21.7
3.43 -, per 1.89 y per 1.89 y per 3.78 7 per 3.78 7 per 1.95 rng.
ml. ml. ml. ml. ml.
Sample Ddta Sheet
volume of the aliquot, was then placed in the cell and a very small amount of pure heptyl alcohol (about 0.01 ml.) added to eliminate foaming. The cell was placed in the constant-temperature bath and the bubbler and electrodes were inserted as diagramed in Figure 1. Wet nitrogen was bubbled through the solution until a constant galvanometer deflection at an applied potential of -0.40 volt us. the saturated calomel electrode w&s secured when the nitrogen bubbler was removed and the gas passed across the surface of the solution. Voltages were set on the instrument, and from the corresponding galvanometer readings, a c-u curve for the sample could be plotted. A method of determinin the amplitude of the c-u curve was selected which did not invofve plotting the curve (see sample data sheet, Figure 3). The galvanometer readings for the limiting current were corrected for the residual current, and the average of a series of amplitudes taken a t 0.025volt intervals starting at an applied otential of -0.27 volt us. the saturated calomel electrode was useaaa a measure of the diffusion current. When t,his average amplitude in microamperes w&s divided by the amplitude per microgram of copper per ml. obtained by calibration, the concentration of copper in micrograms per ml. of the unknown solution was determined. When this concentration was corrected for the volume changes involved in the analysis, the copper concentration in the original copper protein solution was obtained. Dividing this concentration by the dry weight of protein gave the per cent of copper in the dried copper protein. ANALYTICAL DETAILS
EFFECTOF DIALYSISON COPPERPROTEINS.It has been shown by several investigators (7, 8, 9 ) that prolonged dialysis against copper-free water completely removes uncombined or extraneous metallic ions such as those of copper and iron from copper protein solutions. In other words, on dialysis copper protein solutions approach a constant value of copper concentration per milligram of dry weight. If copper ions are added to the copper protein preparation and the preparation is then dialyzed, the copper concentration returns to the original value of the preparation ( 4 ) . I n view of these facts, prolonged dialysis against copper-free water was used as the means of removing extraneous or uncombined copper from all copper protein preparations. CALIBRATION OF TEE DROPPINGMDRCURY ELECTRODE. The instrument was calibrated by adding known amounts of standard
EDITION
251
copper solutions to the dry-weight bulbs and, after following the analytical procedures as outlined, determining the amplitudes of the C-u curves produced. The stock standard copper solutions were made up in two ways, one from copper sulfate and the other from electrolytic copper foil. Capillary 10 was used, having a r n * W ' a constant of 1.58 as determined in the regulating solution. The drop rate was 3.5 seconds er drop9.a determined without any potentirtl being applied. &e temperature was 25.0' C. The two standards proved to have identical concentrations of copper and were used interchangeably. The current which flowed at 25.0" C. in the sodium citrate base solution a t a pH of 4.0 was 0.0624 microampere per microgram of copper per ml. within *0.2% (rn2/at'/* = 1.58, see Table I). DETERMINATION OF RESIDUAL CURRENT. Several investigators, particularly Kolthoff and Lingane ( 9 ) , have emphasized the importance of correcting the amplitude of the c-u curve by that of the residual current. The diffusion currents reported in this paper were all corrected by subtracting the average residual current a t the same voltage. Three experiments, containing no added copper, were averaged and the average curve was estimated to be correct to within ==0.002 microampere. Actual values are tabulated in the sample data sheet (Figure 3). EXTRACTION OF COPPER. That copper can be completely removed from copper proteins by dilute acid extraction, and that no copper becomes adsorbed to the residual protein on subsequently raising the pH to 4.0 in the analytical procedure is shown by Table 11. Experiment 1 was performed by addin a solution containing 1.00 microgram of copper as the cupric sa% to 1.00 ml. of a solution of a high catecholase tyrosinase preparation, C159A. The copper concentration of this cooper protein preparation had been previously determined (see Table 111) as 0.079% copper, dry weight of 1.95 mg. per ml. I n experiments 2 through 7, inclusive, the protein solution was asample of purified ascorbic acid oxidase from which most of the copper had been previously removed by dilute acid dialysis. The sample had a dry weight of 2.53 mg. per ml. and contained 0.014% copper. The experiments in all caaes were performed by adding both copper standard and protein solution to the bulb, evaporating to dryness, and analyzing for copper as outlined in the procedure. r n * h P / ~= 1.58 mg.*/asec.-'/z. Temperature, 25.0" C. The results given in the next to last column show the satisfactory agreement obtained between copper determined by
I.
Cdlibrdtior) of Eloctropode for Microdetermination of Copper hlicroampere Average per y of Experi- Copper Amplitude Cop er Deviation from ment Added Of i d per Mean Mieroampcrs yo dbf Y/ml. Microampcrea 1 60.0 3.12 0.0624 0.0000 0.0 2 5.0 0.313 0.0626 $0.0002 0.3 3 8.0 0.499 0.0624 0.0000 0.0 4 2.0 0.125 0.0628 +0.0004 0.6 6 10.0 0.622 0.0622 -0.0002 0.3 6 1.0 0.0624 0.0000 0.0 0.0624 Mean 0.0624 0.00013 microampere per microgram of copper per ml. Mean deviation = *0.2%.
Table
h.
-
f
Table
II.
Extraction of Copper
Copper Added
Added as in cupric Erperi- Protein ment Added protein ion Ma. ---Microgram1.00 1.54 1 1.95 0.83 5.00 2 2.53 0.17 5.00 3 1.27 0.08 5.00 4 0.63 0.33 52.6 5 2.53 0.17 51.5 6 1.27 0.08 52.1 7 0.63
Total added
Found
2.54 5.33 5.17 5.08 52.9 51.7 52.2
2.58 5.26 5.04 5.02 52.8 52.2 52.7
Deviation from Co er A&d Y
Cu
Approximate Cop er on S r y Weight Bssis
%
S O . 0 4 1.5 -0.07 1.4 -0.132.6 -0.06 1.2 -0.1 0.2 +0.5 1.0 SO.6 1.0
% 0.08 0.2 0.4
0.8 2.0 4.0 8.0
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
252 I1 1.
Reproducibility of Microdetermination of Copper Using the Dropping Mercury Electrode Concentration af Copper D r y Weight Experiment Deternuned of Protein Copper dM 7hE. Mg./ml. % 1.55 2.03 0.076 -0.003 1.50 1.93 0.078 -0.001 1.57 ?.98 0.080 +0.001 I ,98 0.081 +0.002 1.60 1.99 0,080 1.60 +0.001 1.95 0.081 1.58 f0.002 Mean % copper 0.0793 A 0.0016% copper on dry-weight basis. +2%. hIenn deviation
Table
--
analysis and that added to the system both in the combined and ionic form. They likewise show that protein breakdown products that may arise during the preparation of the sample for analysis have no significant effect on the results. LIMITS OF METHOD. The polarographic method of copper assay as developed in this study has a wide range of application, Synthetic copper protein preparations have been satisfactorily analyzed with copper concentrations from 0.08 t o 8.0% copper (see Table 11). Samples of impure naturally occurring copper proteins have been successfully analyzed with as low as 0.005% copper. The actual range in the solution that was analyzed could run as low as 1 microgram per ml. and still achieve a mean deviation within 13%. There was redly no upper limit since the copper protein solution could be diluted, but a practical limit for the copper concentration in the solution to be analyzed was found to be around 75 to 100 micrograms per ml. MEANDEVIATION OF METHOD. A series of analyses was made on a solution of a representative copper protein, tyrosinase, to determine the mean deviation of the method, I n all cases, the dry weight of the copper protein sample was determined and the residue treated as outlined in the analytical procedure, The average of six determinations gave a copper content in the copper protein of 0.0793 * O.OOl6% with a mean deviation of 1 2 % (see Table 111). Comparison of this mean deviation with that generally obtained using the Warburg method shows the polarographic procedure t o be much more precise. These analyses were run on a sample of a high-catecholase tyrosinase preparation, C159A. The copper concentration was determined in all cases as outlined in the procedure. m*/~t'/a = 1.58 mg.'/:sec.-'/,. Temperature, 25.0 "C. Experiment 6 was performed by adding a solution containing 1.OO microgram of copper as the cupric salt to 1.OO ml. of the copper protein in a dry-weight bulb. The total copper present waa determined as 2.58 micrograms per ml. When the copper added as cupric salt was subtracted from the total copper present, the resultin value of 1.58 micrograms per ml. was the amount of copper f u e t o the copper protein present.
INTERFERING IONS.The anaiytical method under discussion was developed primarily for purified copper proteins, and no attempt was made to evaluate critically the influence of other metal ions on the c-v curve of copper. Preliminary studies, however, indicated presence of ferric ion could not be tolerated since the ferric c-u curve was superimposed upon that of copper. INFLUENCE OF HEPTYL ALCOHOL AND NATIVE PROTEIN
A small amount of pure heptyl alcohol was always added tu the cell just prior to the polarographic analysis in order to eliminate frothing during the process of removing dissolved air from the prepared sample by bubbling wet nitrogen through the cell contents. Heptyl alcohol has a solubility a t 25.0" C. of 0,181 gram per 100 grams of saturated solution ( I ) or about 0.27,. Thus the addition of 0.01 ml. of the alcohol (about 2%) to the sample (volume 0.55 ml.) was in actuality far in excess of its solubility. For this reason it was not necessary to correct the volume of the solution for the alcohol added. The excellent
Vol. 17, No. 4
agreement between copper added and copper found (see Tables I1 and IV) shows that the heptyl alcohol had no significant effect on the amplitude of the c-u curve over a considerable range of copper concentration. I n the procedure as outlined, the copper protein is evaporated to dryness and then extracted with acid to remove the copper. Although one would expect the major portion of the protein to be denatured by this treatment, it seemed advisable to determine what amount of native protein could be tolerated in the sample during the polarographic analysis. For this purpose a series of experiments was performed wherein varying amounts of native protein egg albumin and hemoglobin were added to sodium citrate-fuchsin-copper solutions of known copper concentration just prior to the polarographic analysis-Le., the samples were not evaporated to dryness and extracted with acid. The results are given in Table IV, and some experimental details are given below. All experiments (Table IV) were performed by adding 0.00570 fuchsin and 2% n-heptyl alcohol to a base solution of acid sodium citrate buffer and, after adding the solution of native protein and the alcohol, polarographing manually as described in the procedure. I n experiments 2 3, and 4 a solution of egg albumin. prepared by the method 0 ) S6rensen and HZyrup ( 2 4 , waa used 89 the protein. It had been rendered copper-free by prolonged dialysis against copper-free water. I n experiment 6 a solution of hemoglobin, prepared as described by Schmidt (25), was used, which had been likewise dialyzed against copper-free water. The heptyl alcohol was an Eastman Kodak preparation and was not purified further. VL*/JPi s = 1.58 mg.*/1sec.-'/2. Temperature, 25.0' C.
IV.
Influence of Native Protein on Copper Wave Formed in a Sodium Citrate Base Solution with A d d e d Fuchsin Concentration Experiment Protein Added of Copper Found Deviation Copper Added from
Table
70
r/ml.
r/mL
-t/ml.
%d
0.00 0.06 0.12 0.24 0.00 0.08
25.4 25.2 24.9 24.5 0.0 0.0
25.4 25.4 24.7 24.1 -0.1
0 0 +o 2 -0 2 -0 4 -0 1 -0.1
0.0 0.8 0.8
-0.1
1.6
... ...
The fact that there was no interference due to iron, in the case
of the hemoglobin, would indicate that no free iron was liberated from the heme-protein a t the pH of the analytical solution (pH 4.0). In a typical analysis, as shown in Figure 3, about 2 mg. of copper protein are involved. After the extraction, the maximum possible protein concentration in the sample is in the order of 0.370, and as previously pointed out, this concentration would not be expected because of the denaturation occurring during the preparation of the sample. For this reason the amounts of native protein used in the experiments of Table I V were adequate to test the native protein tolerance of the c-v curve. The results show that the deviation caused by the addition of native protein to the extent of 0.25y0 was less than 3=2% and therefore fel! within the experimental error of the method. There was no significant deviation in the residual current. CALCULATION OF THE HALF-WAVE POTENTIAL
The half-wave potential, ,TI/*, of copper in acidsodium citrate solution containing 0.005% fuchsin has been approximated by Reed and Cummings (23) as -0.15 volt us. the saturated calomel electrode. However, if the origins in the polarograms given by these investigators are comparable, as is indicated, inspection indicates El,*values ranging from -0.14 t o -0.23 volt us. the saturated calomel electrode, which can scarcely be accounted for by the lack of temperature control. In view of this variation, the half-wave potential was redetermined on the basis of the data obtained in this investigation. The average El, 2 us. the anode potential was determined Tithin +0.01 volt as -0.31 volt for four c-v curves selected at random. The anode potential u . the saturated calomel electrode was carefully determined within
ANALYTICAL EDITION
April, 1945
253
seems of interest therefore to Tompare this value with that obtained in the sodium citrate buffer ( p H 4) used in this study. From the data in Table I it can be calculated that the diffusion current constant (15) = id/(Cm''st'/') = 2.51. Therefore, D = 0.43 X cm.z sec.-l. APPLICATIONS
I n the field of copper proteins, the polarographic method for the niicrodetermination of copper has been shown t o be applicable for varied preparations. Tyrosinase, ascorbic acid oxidase, hemocyanin, and the copper protein in the stroma of erythrocytes have been satisfactorily analyzed for copper by the dropping mercury electrode. I n cases where simultaneous deterniinations were made, the results obtained by the polarographic procedure were comparable with those obtained by other methods, both manometric and colorimetric. ACKNOWLEDGMENT
-0.05 - 0 l O
-0.15
-020
- 0 . 2 5 -030 -035
.Eae,vs. S.C€ , V o l t s Figure 4.
c-v Curve of Co per in A c i d Sodium Citrate Base folution
The curye was obtained by polarographic analysis of an acid sodium citrate solution which was l o - ' M in COP er (6.36 micrograms of copper per ml.) and 0.005% in fuchsin. The amplitudb d the curve obtained was corrected for the residual current. Experimental details are siven in the analytical procedure. ma/al'/a 1.58 mg.'/' sec.-'/l. Temperature 25.0' C.
-
-
*0.001 volt as +0.126 volt. Thus for copper in the above base solution, the half-wave potential was determined within +0.01 volt as -0.18 volt us. the saturated calomel electrode (see Figure 4 ) . Since the anode potential is constant for a given base solution, this measurement was omitted in routine analyses.
The authors wish to express appreciation t o J. J. Beaver of Columbia University and to J. J. Lingane of Harvard Lniversity for helpful advice and criticism. Appreciation is expressed to Stanley LeKis of these laboratories who prepared the tyrosinase and ascorbic acid oxidase preparations (Table 11). LITERATURE CITED
Butler, J. A . V., Thoinson, D. IT., and Maclennen, W. H., J . Chem. SOC.(London), 1933, I, 674. (2) Coulson, E. J.. J . Assoc. Official Agr. Chem., 20, 179 (1937). (3) Dalton, H. R., and Xelson, J . M.,J . Am. Chem. Soc., 61, 2946 (1)
(1939). (4)
(5) (6)
T H E ELECTRODE REACTlON
(7)
A comparison of the amplitude of the copper c.u curve with those of other ions, whose elect'rode reactions are known, indicates that cupric ion in an acid sodium citrate solution behaves like such bivalent ions as cadmium, zinc, and lead. The single wave (Figure 4),obtained whether increasing or decreasing the applied potential, is the result of a reversible two-electron reduction of the citrate cupric complex t o metallic copper at the cathode. The value of E 3 l 4 - E l l r (Figure 4) is 0.03 volt, which is the ex-
(8)
?):'(
pected value
for a reversible two-electron reaction
( 1 4 ) . The reversible character of the reaction is also verified by the fact that a straight line is obtained by plotting log (i/id - i) us. Ed..,. I n high concentrations of copper, the small wave due to fuchsin at about -0.72 volt us. the saturated calomel electrode is barely discernible, and no further wave is observed until around - 1.7 volts vs. the saturated calomel electrode at the decomposition potential of sodium ion (or citrate ion). These results correspond with those of Kolthoff and Lingane (11) who observed a single wave for copper in tartrate medium, the height of which corresponded t o Cu'+ + Cu(Hg). A41thoughinformation regarding the nature of the citrate cupric complex could have been obtained by measuring the half-wave potential BS a function of the concentration of citrate ion, no such study was attempted in this investigation. From titration curves Smythe (27) indicates that it is a complex basic salt in which the hydroxyl group enters into combination in a manner similar to that which he postulates for the cupric tartrate complex. Murata (20) indicates several of the different structural configurations possible for a complex compound of this type. THE D I F F U S I O N COEFFICIENT
Lingane (15) found the diffusion coefficient, D, for cupric ion in acid tart,rate solutions to be 0.39 X sec.-l. I t
(9)
Dills, W.L., and Kelson, J . l l . , Ibid., 64, 1616 (1942). Dixon. M., "Manometric Methods", 2nd ed., Chap. 11, Cambridge, University Press, 1943. Eden, rl., and Green, H. H., Biochem. J . . 34, 1202 (1940). Green, D. E., "Mechanisms of Biological Oxidations", p. 107, Cambridge, University Press, 1940. Kamegai, S.,J . Biochem. (Japan),29, 439 (1939). Kolthoff, I. M.,and Lingane, J. J . , "Polarographic .Inalysia and Voltammetry Amperometric Titrations", pp. 111-1 12. New York. Interscience Publishers, 1941.
(10) I i d . , p. 244. (11) I h i d . , p. 280. (12) Kubowita, F., Biochem. Z., 292, 221 (1937). (13) Latimer, W.hl., and Hildebrand, J . H., "Reference Book of Inorganic Chemistry", p . 105, K'ew York, Maemillan Co. 1940. (14) Lingane, J . J., Chem. Revs., 29, 1 (1941). (15) Lingane, J. J., IND.ESG.CHEM.,ANAL.ED.,15, 583 (1943). (IC,) Lovett-Janison, P. L., and Nelson, J. M., J . Am. Chem. Soc., 62, 1409 (1940). (17) Ludwig, B. J., and Nelson, J . M., Ibid., 61,2601 (1939). (18) Mandai, H., Acta Schol. .Ired. Univ. Imp. Kioto, 14, 167 (1931). (191 Mann, T., and Keilin, D., Proc. R o y . Soc., B, 126, 303 (1938). 120) !vfurata, K. J., J . Wash. Acad. Sci., 27, 101 (1937). (21) P:w!iinson. G. G., Jr., and K'elaon, J. M., J . Am. Chem. Soc., 62, 1693 (1940).
( 2 2 ) Powers, W.H., Lewis, S . , and Dawson, C. R., J . Gen. Physiol., 27. ~,~~167 11944). j
~
~
,
f23)
Reed, J . F., and Curnmings, R. IT., IXD.ESG. CHEM.,A N ~ L .
(24)
Roncato, A . , and Bassani, B., Arch. sei. bid. (Italy), 19, 541
ED.,12, 489 (1940). (1934).
(25) Schmidt, C. L. A,, "Chemistry of the Amino Acids and Pro.
teins", p. 176, Springfield, Ill., Charles C . Thomas Co., 1938. (26) Shoji, K., Bull. Inst. Phys. Chem. Research ( T o k y o ) , 10, 162 (1931).
( 2 7 ) Smythe, C. V., J . B i d . Chem., 92, 233 (1931). ( 2 s ) SGrensen. S . P. L., and Hgyrup, M.,Compt. rend. lab. Carlsberg, 12, 12 (1917). (29) Stotz, E., J . B i d . Chem., 133, c (1940). (30) Stout, P. R., Levy, J., and Williams, L. C., Collection Czechoaiop. Chem. Commun., 10, 129 (1938). (31) Such,,, K., Ibid., 3, 354 (1931). (32) Thanheiser, G., and Maassen, G., Arch. Eisenhuttenu., 10, 441 (1937). (:13) \Tarburg, O., Biochem. Z., 187, 255 (1927). (.'14) Warburg, O., and Krebs, H . A . , l b i d . , 190 143 (1927)
