Qu a ntita tive Determina ti on of Cysteic Acid in Protein Hydrolyzates Rapid Paper Electrophoretic Method J. F. DlEHL
Department o f Biochemistry, School of Medicine, Universiiy of Arkansas, Little Rock, Ark. ,A simple and ropid method for the determination of cysteic acid in protein hydrolyzates was developed, using low-voltage p a p e r electrophoresis for complete separation of cysteic acid from other o m i n o acids a t pH 3.6. Two procedures ore described for the quontitative determination of the cysteic acid after color reaction with ninhydrin in the presence of cupric ionsone using direct photometry of the p a p e r strip, the other elution ond colorimetry. Both procedures hove been applied successfully to the determination of the cysteic acid content of industrial wool specimens.
A
capable of determining as little as 0.05'% cysteic acid in proteins, using high-voltage paper electrophoresis for the separation of cysteic acid from the other amino acids of the protein hydrolysate, was recently described by Zuber, Ziegler, and Zahn (6). This procedure requires expensive npparatus, which is not available in many laboratories. The present paper describes the use of a simple low-voltage paper electrophoresis cell for the separation of the cysteic acid from the other components of protein hydrolye a k s and quantitative determination by reaction with ninhydrin followed by direct photometry on paper or by elution and colorimetry. It can also be applied t o the determination of cystine plus cysteine after oxidation to cysteic acid according to Schram, Moore, and Bigwood (4) and Znber, Ziegler, and Zahn (7). METHOD
EXPERIMENTAL
Electroohoresis. Electronhoretic separations were carried out in a Durrum-type paper electrophoresis cell (Spinco Model R) operated by a Spinco Duostat power supply. [For a description of the Spinco Model R Paper Electrophoresis System see Block. Durrum, and Zweig ( I ) ] . The cell accommodates eieht Daner strim 31.5 em. long and 2.9 e.; in' Gidth. %h'am t an 3 MM paper strips were used. The pH 3.6 buffer (5) was prepared by mixing 1 volume of pyridine with 10 volumes of
1204
ANALYTICAL CHEMISTRY
glacial acetic acid and 89 volumes of water. For each run, eight strips were marked with pencil, hung like an inverted V over the rack in the electrophoresis cell and soaked by pouring buffer over the strips along their apices. The strips were permitted to drain for 20 minutes. After this time the cysteic acid solutions or protein bydrolyeates were evenly streaked across the paper strips at the apices by means of micropipets. A very even distribution of the solutions across the width of the strips was found to be particularly important when the strips were to be evaluated by direct scanning. Six and 10 PI. of a standard cysteic acid solution containing 1mg. of cysteic acid per ml. were applied to two strips, leaving six strips for the evaluation of test solutions. Electrophoresis was allowed to proceed for 2 hours at a constant current of 30 ma. The voltage was about 280 volts at the beginning of a run and about 230 volts after 2 hours. This represents a voltage gradient of 8.9 and 7.3 volts per em., respectively. Upon completion of a run, the rack with the strips was taken from the electrophoresis cell, extended to bring the strips in horizontal position, and placed for 15 minutes in an oven set a t 105" C . Color Development. The dried strips were simultaneously immersed for 10 seconds in a solution of 5 grams of ninhydrin in a mixture of 930 ml. of 1-butanol and 70 ml. of glacial acetic acid, using the staining rack and tray provided with the Spinco electrophoresis apparatus. When the excess liquid had dripped off the strips, they were transferred with forceps from the staining rack to a drying rack and heated for 15minutes in an oven at 6OOC. Full color development was brought about by either saturating the atmosphere in the oven with water or by holding the rack with the strips over a steam bath for 30 seconds after the heat treatment in a dry oven. The strips were then carefully sprayed from both sides with a solution of copper nitrate in ethyl alcohol, as proposed by Wieland and Kawerau (6). This treatment, which results in greater stability of the color and higher sensitivity of the quantitative determination, causes a color change from blue to pink. Direct Photometry. After a 10minute interval, t o allow for evapora-
Figure 1. Electrophoretic seporation of cysteic acid Cysteic acid alone Artificial mixture of cysteic acid with 18 other omin0 acids C. Hydroiyrote of wool ( O . Z Z ~ o cyrteic m i d on dry wool basid Electrophoresis conditione 30 ma.. 280 to 230 volts, 2 hours, pH 3.6. White line indicate. where mmples were applied A.
B.
tion of the ethyl alcohol, the cysteic acid hands of the two standard strips and the six t p s t strips were scanned in a balance beam type of photoelectric recording photometer (Spinco Analytrol Model R). This instrument produces a record of the variations of absorbance along the strip when the B-2 balancing cam is used. The integrated areas under the density peaks are given in square centimeters (X IO-l). A slit width of 1 mm. was used, together with 500-mp interference filters. A standard curve was prepared by plot.ting the two known concentrations against the densities and drawing a straight line from the
I
Figure 2. Relationship between cysteic acid concentration and absorbance
A
i
200
.2
150 z 4
t'I
100
g t
c
k
1 /.;7
.I00
A-COLORIMETRY of ELUATES 0-DIRECT PHOTOMETRY
50
3
!k
i
2 4 6 8 IO 12 20 8 CYSTEIC ACID PER PAPER STRIP
origin to the two points thus obtained or, if the two points did not exactly fall on a straight line with the origin, a straight line was drawn from the origin, approaching the two points equally closely. The concentrations of cysteic acid on the other six strips m-ere calculated by reference to this standard curve. A new standard curve must be prepared for every run, as the absorbances are not well reproducible in repeated runs. Elution and Colorimetry. The cysteic acid concentrations mere also determined by an elution procedure. The pink bands in the cysteic acid position were cut out, placed in test tubes, and extracted with 4 ml. of 95% ethyl alcohol each. I n addition t o the cysteic acid bands on the eight paper strips, one piece of the same area was cut from a nonstained part of one of the eight strips and extracted in the same manner, to serve as a blank. One-half hour, with frequent shaking of the test tubes, was allowed for the extraction. The absorbances were then determined in a Beckman DU spectrophotometer a t 504 mp. Occasionally a Coleman Junior spectrophotometer was used. The results obtained with the two strips which carried known amounts of cysteic acid were plotted on graph paper and a standard curve (straight line from origin) was drawn. The concentration of cysteic acid on the other six strips n-as determined from the standard curve. As with the direct photometry procedure, a new standard curve must be prepared for every run. Preparation of Protein Hydrolyzates. Samples of wool, roughly 1 gram in weight, were dried for 2 hours in an oven a t 105' C. The cooled samples were accurately weighed in closed weighing glasses and transferred t o test tubes. The tubes were sealed after adding 10 ml. of 1 to 1 hydrochloric acid, and were heated for 24 hours in an oven a t 105" C. They were opened after cooling to room temperature and their contents were evaporated in vacuo. Evaporation was repeated three times with 15-ml. portions of water in order t o remove most of the hydrochloric acid. The residues were dissolved in a few milliliters of water and the volume was made up to 10 ml.
in a volumetric flask. When it was found that a wool sample contained less than 0.2% of cysteic acid, the hydrolysis was repeated and the hydrolyzate was made up to a volume of only 5 ml. When hydrolysates of unknown cysteic acid content were to be analyzed, three samples of 2, 10, and 20 111. were applied to the paper strip. When the cysteic acid content was roughly known, the volumes applied for analysis were adjusted in such a way that the cysteic acid concentration per paper strip ranged from 4 to 20 y when the elution procedure was to be used, and 4 to 12 y for direct photometry.
Table t. Accuracy and Precision of Cysteic Acid Determinations by LOWVoltage Method
Calcd. Concn., Mg./100 MI. Direct Elut,ion scanning 84.5 83.1 119.0 80.3 84.5 77.5 66.5 92.7 71.9 77.5 94.0 86.4 65.1 81.2 66.2 83.1 65.3 85.6 Av. 83.0 79.7 Dev. from theoretical value, 84.6 mg./100 ml., 70 5.8 1.9 Std. dev.", mg./100 ml. 18.1 4.8 d = difference between individual
result and average N = number of determinations
Table II.
Recovery of Cysteic Acid
Cysteic Acid Added,
Found,
Recovered,
Y
Y
%
0 2
5.0 6.7 9.0 10.8
...
4 6
96 100 97
RESULTS A N D DISCUSSION
Figure 1 shows the separation of cysteic acid from a mixture with 18 other amino acids commonly occurring in acid protein hydrolyzates. Another strip shows the separation of cysteic acid from the other amino acids of a wool hydrolyzate. The photograph was made after color development with ninhydrin and before spraying with copper nitrate solution. Figure 2 shows that the results of the elution procedure follow Beer's law over a much wider range than those obtained by direct photometry. Absorbances measured in the spectrophotometer fall on a straight line for cysteic acid quantities up to 20 y, while the results obtained by direct photometry approximate the straight-line relationship only for cysteic acid quantities up to 12 y . For both procedures 2 y of cysteic acid per paper strip appeared to be the lower limit for accurate determination. The accuracy and precision of both procedures were established by determining the cysteic acid content of a solution of 19 amino acids commonly occurring in acid protein hydrolysates. One-half millimole of cysteic acid (84.6 mg.) and 1 millimole of each of the other 18 amino acids were dissolved in water plus a few drops of concentrated hydrochloric acid, making a total vol-
ume of 100 ml. Of this solution volumes ranging from 4 to 14 pl. were applied to nine paper strips and analyzed for cysteic acid, The average values of 83.0 and 79.7 mg. (Table I) differed from the theoretical value by 1.9% for the elution procedure and 5.8% for the direct photometry procedure. The standard deviations, 4.8 and 18.1 mg. per 100 ml., respectively, indicate that the elution procedure can be repeated with good precision and the direct photometry with a precision satisfactory for many purposes. In addition to the elution procedure and the direct scanning described in the experimental part, the author has evaluated the strips from a number of electrophoresis runs by other ninhydrin methods. The procedure published by Giri, Radhakrishnan, and Vaidyanathan (a), using a very dilute alcoholic solution of cupric ions for the elution of the ninhydrin colored amino acid bands gave essentially the same precision and accuracy as the Weland and Kawerau method described above. Two other procedures, using ninhydrin without the addition of cupric ions gave unsatisfactory results. The recovery of cysteic acid in the complete procedure was determined by hydrolyzing four I-gram samples of bleached wool top together with added VOL. 31, NO. 7, JULY 1959
1205
Table 111.
Cysteic Acid Analyses of Commercial Wool Samples
Average values of at least three determinations Mg. Cysteic acid per Gram Wool ~ Dry _ ___ _ HighDirect Wool Samples photomvoltage Description etry Elution method 2 2 2 2 2 5 Kool top, unbleached 18 5 0 6 3 Wool top, peroxide bleached Australian wool, solvent 1 7 1 3 1 3 extracted only 17.0 15 8 15 5 Texas mohair, treatment unknown Plain weave fabric ( 3 2 / 2 ) , 11 .o 90 10 0 shrink-proofed by chlorination
SO.
1 2 3 4
3
amounts of 0, 2, 4, and 6 mg. of cysteic acid (Table 11). The observation by Schram et al. (4)that practically no cystcic acid is lost under the conditions of acid hydrolysis of proteins is confirmed. Table I11 shows the results obtained with five actual protein hydrolysates as compared with those obtained by Klaus Ziegler of the German Wool Research Institute, who analyzed samples of the same wool specimens, using the highvoltage technique (6). The results obtained by the two procedures described in this paper compare favorably with those obtained by the highvoltage method. As expected, the cysteic acid contents of the oxidized specimens 2 and 5 are much higher than those of untreated samples 1 and 3. CONCLUSIONS
The present investigation shows that cysteic acid can be completely separated from the other amino acids of a protein hydrolyzate by low-voltage paper elec-
trophoresis. Quantitatiyc determination is possible either b!. direct photometry or by colorimetry after elution. Applied to industrial wool svmples the method has shown that blenching or shrink-proofing treatments can increase the cysteic acid content of ivool fivefold and more. When a protein hydrolyzate is ready for analysis, a skilled technician can carry out the cysteic acid determination by the method here described within 5.5 hours following the elution procedure or 4.5 hours using direct scanning. T o increase accuracy, two or three strips should be used for each hydrolyzate. As two of the eight strips are required for construction of the standard curve, two or three different hydrolysates can be analyzed simultaneously within this period of time. This is hardly possible with paper chromatography or ion exchange methods. For research purposes the more accurate elution procedure is preferable to the faster method of direct scanning, while for certain industrial routine determinations of
cysteic acid the latter method would appear satisfactory. Low-voltage paper electrophoresis promises to become a particularly valuable tool in the control laboratories of the ~voolindustry. ACKNOWLEDGMENT
The author is much indebted to Helmut Zahn. Director of the German n 7 ~ 0Research 1 Institute a t Aachen, for making available the results of cysteic acid analyses carried out in his institute, and for supplying samples of the same wool specimens analyzed by his group. The Xnalytrol readings have been obtained through the kind cooperation of James E. Dusenberry of the Universitv of Arkansas School of Pharmacy. The technical assistance of Margaret Ashcraft is gratefully acknowledged. LITERATURE CITED
(1) Block, R. J., Durrum, E. L., Zweig, G., ‘‘Manual of Paper Chromatography and Paper Electrophoresis,” 2nd ed., p. 525, Academic Press, New York, 1958. (2) Giri, K. V., Radhakrishnan, A. X., Vaidyanathan, C. S., Nature 170, 1025 (1952). (3) Ryle, A. P., Sanger, F., Smith, L. F., Kitai, R., Biochem. J. 60, 541 (1955). (4) Schram, E., Moore, S., Bigxood, E. J., Ibid., 57, 33 (1954). (5) Wieland, T., Kawerau, E., Nature 168, 77 (1951). (6) Zuber, H., Ziegler, K., Zahn, H., 2. Naturforsch. 12b, 531 (1957). (7) Ibid.,p. 734. RECEIVEDfor review December 1, 1958. Accepted February 20, 1959. Work s u p ported by Grant A-721(ClO) from the National Institutes of Health. Public Health Service.
Gas and Liquid Elution Chromatography Quantitative Detector Evaluation H. W. JOHNSON,
Jr., and
F. H. STROSS
Shell Development Co., Emeryville, Calif.
b Quantitative methods for adequately evaluating detectors have not been available. This paper discusses desirable detector properties and proposes a specific evaluation procedure. A quantitative treatment of noise is developed, making possible a precise statistical definition for the limit of detection.
G
chromatography (GLC) is widely used to analyze a variety of mixtures. Yevertheless, AS-LIQUID
1206
ANALYTICAL CHEMISTRY
its applicability could be extended by improving apparatus performance. Many attempts to improve the detector, the device which analyzes the gas stream emerging from a column, and several detector designs have been reported. Cnfortunately, there is no standard method of describing detectors quantitatively, and it is difficult to compare performance. The same problem will arise in liquid chromatography as suitable detectors are developed. This paper deals mainly with the
estimation of the smallest amount of sample that can ‘be determined reliably by a detector. In the past, detector sensitivity has often been used as a measure of this quantity. This is unwarranted because a detector with high sensitivity cannot necessarily detect very small samples. Sensitivity was defined for gas chromatography as
