404
V O L U M E 19, NO. 6
detecting the small traces of carbon dioxide which go to make up the over-all blank. By checking and making modifications in t h e procedure, the authors were able to reduce the over-all blank t o a reproducible minimum. To test the over-all precision, reproducibility, and accuracy of the method, the authors used two carefully prepared samples, one a carbon steel from the Bureau of Standards and the other a high-silicon steel from the American Rolling Mill Company. Portions of each were sent to four laboratories using the lowpressure method. T o determine whether the apparatus and procedure were applicable to other steels, some samples of stainless, heat-resistant, and corrosion-resistant steels were analyzed. Results of analysis of these steels reported in Table I indicatct t h a t the low-pressure gasonietric method gives results which compare favorably in reproducibility and accuracy n ith thc jesults obtained by other low-pressure niethods.
ACKhOWLEDGRlEYT
The authors gratefully acknowledge the assistance of Guy Burrell and Lloyd Guild of Burrell Technical Supply Company, Pittsburgh, Pa., in supplying special items of glassware required for the experimental work. LITERATURE CITED
(1) Murray, W. M.,and Ashley, S. E. Q., IND.E m . CHEM.,AKAL. E D . , 16, 242 (1944). (2) Stanley, J. K., and Yensen, T. D., Ibid., 17, 699 (1945). (8) U. S. Steel Corp. Chemists, "Sampling and Analysis of Carbon and Alloy Steels,:' Yew York, Reinhold Publishing Corp., 1938. (4) Wooten, L. A, and Guldner, W.G., IXD. ENG.C H E X . ,X h - a ~ . E D . , 14,835 (1942). (5) Yensen, T. D., Trans. A m . Electrochem. Soc., 37, 2 2 i (1920). (6) Ziegler. ?;. A , , I h i d . , 56, 231 (1929).
Determination of Cystine and Cysteine in Altered Human Hair Fibers DOROTHT SANFORD 4 Y D FRED L. HUJIOLLER, Rexenrrh Division, R a y m o n d Laboratories, Znc., S t . P a u l , M i n n .
To determine the amount of cystine in altered human hair in the presence of cysteine, part of the hair is hydrolyzed with 1 to 1 hydrochloric acid for 6 hours at 118" to 120" C. in a closed tube, and the total cysteine plus cystine is determined by the Sullivan method. Another sample is alkylated with the least exposure' to air, using an excess of 1% iodoacetate at pH 8.3, at the temperature of the
0
F THE several methods available for the quantitative esti-
mation of cystine in unaltered human and animal hair fibers, the Sullivan (S), Okuda ( 6 ) , Folin and Looney ( 2 ) , and Brdgcka polarographic (1) methods and their modifications are most frequently used. In the work reported here the Sullivan colorimetric method was used exclusively. To be of any value in the study of the fundamental chemistry of hair waving by the so-called permanent methods, a n i method for the quantitative estimation of cystine must be capable of being modified in such a manner that cystine can be determined in the presence of cysteine. This is necessary in order to follow the degree of reduction produced by the e\periniental alteration of the hair. I n all quantitative cystine methods it is necessary t o hydrolyze the hair with acid for several hours in order t o break the keratin down into its amino acid constituents. Obviously, such treatment may lead to the iroxidation of somt' of the cysteine formed in the experiment if hydrochloric or sulfuric acids are used for hydrolysis, for hair which ha? hcrn reduced by commercial waving solutions always contains traces of heavy metals such as iron or copper and these are efficient catalysts for the reoxidation of cysteine. Since thorough vashing after the reduction step, in the authors' experience, will lead to considerable oxidation of cysteine, the metallic impurities will be carried over into the final hydrolysis mixture. On the other hand, if hydriodic acid is used in the hydrolysis, all cystine is reduced to cysteine. Sullivan, Hess, and Howard (9) have published a method for the estimation of cystine and cysteine in mixtures of these two amino acids and applied it to purified proteins (3). A careful study of their method as well as of the practical problem of analyziyg
boiling water bath for 30 minutes. The hair is then dried, chopped into small pieces, and hydrolyzed, and the amount of residual cystine is determined by the Sullivan method. Subtracting the latter values from the former gives the amount of cysteine formed. Studies with cold waving reducing and oxidizing solutions show that at room temperature reactions are practically complete within 4 minutes.
altered human hair for these two amino acids in the presence of each other convinced the authors that it was not suitable for their purpose and hence was not tried. Since hydrolysis of the hair is the first step in the determination of cystine, a study was carried out to determine the optimum conditions of hydrolysis-that is, that condition which would cause a minimum amount of destruction of either cystine or cysteine. The open flask method of hydrolysis using 1 to 1 hydrochloric acid, a mixture of formic and hydrochloric acids as recommended by Miller and Du Vigneaud ( 4 ) , or constantboiling hydriodic acid failed to give cystine values in this laboratory for unmodified hair which are consistent with those reported in the literature. Therefore, a study of the sealed tube technique of hydrolysis was undertaken.
T o this end, about 50-mg. samples of accurately weighed hair which had been dried in an oven a t 110' C. for 2 hours were placed in Pyrex test tubes, 5 ml. of 1 to 1 hydrochloric acid (prepared by diluting commercial C.P. hydrochloric acid with an equal volume of water) were added, and the tubes were sealed. In order to prevent accidents, each glass tube was placed inside a piece of gas pipe, closed by screwing caps on both ends. These tubes were then placed in an oven set at 118" to 120" C. for varying lengths of time. After the heating period, the tubes were allowed to cool and opened. The contents were then quantitatively transferred to a 100-ml. beaker and the excess hydrochloric acid was neutralized t o pH 3.5 by the droptvise addition of 5 8 sodium hydroxide; a glass electrode was used to detect the end point. The solutions were then diluted to 100 ml. with a solution of hydrochloric acid adjusted to pH 3.5 and the amount of cystine was determined on an aliquot by the Sullivan method.
405
JUNE 1947 Table I . Effect of Time upon Degree of Hydrolysis of Human Hair as Determined by Amount of Cystine Liberated Time of Hydrolysis Hours 2
4 5 6 7 8 9 12 1A
20
Cystine Found
Average
7%
% 12.7 16.4 16.6 16.5 16.1 16.6 15.3 15.8 15.1 15 4
13.1,12.8,12.3 16,5,16.2,16.4 16.5,16.6,16.7 16.2,16,7,16.5 16.1,15.7,16.4 16.7,16.5,16.5 15.6,15.2,15.3 15.9,15.6 15 5.14 7 15 5,15 2
Table 11. Effect of Time upon Degree of Hydroljsis of Wool as Determined by .&mount of Cystine Liberated Time of Hydrolysis Hours 2 4 6 7 8
___
~ _ _ _ ~
Cystine Found
R 7 9 9 9 9 .
9 6 9 7 6 - --
Table I list’s the results obtained on unmodified human hair, and Table I1 gives the results obtained in the hydrolysis of a sample of sheep’s wool. These results show t,hat. under the conditions of these experiments-that is, a t 118’ to 120” C. and using 1 to 1 hydrochloric acid for hydrolysis-a heating period from 4 to 8 hours gives maximum recovery. For routine work in this laboratory a 6-hour period of hydrolysis has been adopted. In order to determine cystine in the presence of cysteine, it is necessary to treat the hair in such a manner that. either the cysteine or the cyst,ine no longer produces a color wit,h Sullivan’s reagent whereas the other still will react quantitatively. Harris (7) and his collaborators have used et,hylbromide for this purpose.
h sample of reduced hair or wool (0.5 gram) is suspended in a phosphate buffer solution of pH 8.3 and 0.5 ml. of ethyl bromide is added. The sample is shaken for 18 to 20 hours, washed with water, and then hydrolyzed as usual. Ethyl bromide reacts with sulfhydryl groups to form ethyl sulfides according to the equation R-SH
+ BrCH2CHB+ R-S-CZHs
+ HRr
These sulfides or thioethers are stable to hydrolysis with 1 to 1 hydrochloric acid and no longer produce a color with Sullivan’s reagent. Cystine does not react with ethyl bromide under these condition?. This method, however, requires 18 to 20 hours for the alkylation step alone; ivhere many samples are to be run it would br :idvantageous if this time could be reduced. To t,hat end other halides were investigat,ed. Since in enzyme chemistry iodoacetic wid is used effectively t,o block -SH groups, this reagent was also investigated. Mirsky and ;inson ( 6 ) also used iodoacetate to block -SH groups in their modification of the Folin phosphotungstic acid method for cystine in the presence of cysteine. Preliminary experiments indicated that the reaction bctneen iodoacetic acid and the sulfhydryl groups of reduced hair is relatively slon- at room temperature: hence, hrating n-as resorted to. In a typical experiment, a sample of hair was reduced in an excess of a commercial cold waving solution which was 0.72 ,V in thioglycolate and had a pH of 9.2, the alkalinity being due to excess ammonium hydroxide. The hair was immersed in this solution for 15 minutes, then washed first in 10% ammonium hydroxide and then in tap water. After being dried in a vacuum desiccator it was chopped into small pieces. Part of the reduced hair was alkylated by the Harris method (shaking with ethyl bromide in a phosphate buffer a t pH 8.3 for 20 hours), while in another part of the chopped hair the sulfhydryl groups were blocked by heating in a 1% solution of iodoacetic acid made alkaline to pH 8.3 by the addition of sodium carbonate for 15 and for
90 minutes. The two samples of hair were then washed, dried, and analyzed for their residual cystine content by the Sullivan method. The results listed in Table I11 seem to indicate that with a reduction period of 15 minutes in alkaline ammonium t,hioglycwlate, about one half of t,he cystine is reduced to cysteine, and that an “alkylation” of 15 minutes with iodoacet.ate is as c+dvc alkylation for 18 t8020 hours with et,hyl broniidc. Since there remained some doubt as to the amount of reoxidation of the hair during the process of drying, a sample of the same hair used in the previous experiment was reduced with a solution of ammonium thioglycolate of the same strength and pH, but the time of reduction was cut to 4 minutes in one case and to 8 minutes in another case. The hair samples were removed from the reducing solution and washed no longer than 30 seconds in water (in the case of the hair to be alkylated Tvith ethyl bromide) or in an ice-cold 1% solution of iodoacetic acid (in the case of the hair to be “alkylated” n.ith iodoacetate), The hair samples were then transferred to their respective alkylating solutions and after having been alkylated as indicated in Table I V were washed, dried, and analyzed for their residual cystine content as before The results of t,his experiment, which is typical of many others carried out, show the importance of not drying the hair previour to alkylation, as otherwise a large percentage of the cystrinc, formed in the reduction step is reoxidized t’o cystine. The met.hod of analysis which has been adopted for routinc work in this laboratory is the outcome of theie studies.
A tress of hair, usually weighing about 200 mg., is reduced. It is then quickly transferred to a cold 1% solution of iodoacetic acid contained in a beaker. After being washed in this solution for about 1 minute it is stripped free of excess liquid (rubber gloves should be worn for this step) and quickly transferred to a large test tube containing about 50 ml. of a 1% alkaline solution of sodium iodoacetate, pH 8.3, heated in a boiling water bath. The hair is allowed to react for 30 minutes n.hile the bath is kept boiling, then removed, mashed thoroughly in water, and dried a t room temperature. The hair is then chopped into small pieces by either an electric clipper or a pair of scissors, and dried for 2 hours a t 110’ C. From 50- to 100-mg. samples of the chopped hair are weighed off in small glass cups, made by cutting off the ends of small homeopathic vials, and these in turn are dropped into 15 x 125 mm. Pyrex test tubes containing 5 ml. of 1 to 1 hydrochloric acid. The tubes are sealed and the hair is hydrolyzed for6 hours a t 118’ to 120 a C. The amount of residual cystine is then determined by the Sullivan method on aliquots as outlined before. Along with the sample of hair which has been modified, another sample of the same hair is hydrolyzed without carrying it through the reduction step. This latter sample will give the total cystine
Table 111. Cystine Content of Hair Reduced w - i t h 0.72 .V Thioglycolate, pH 9.2 Cystine Found, Treatment of Hair Control, unreduced Blocked with ethyl bromide Blocked with iodoacetic acid, 15 minutes Blocked with iodoacetir arid. 90 minutes
7%
16.3 16.3
8.2 8.1 8.4 8.0 8 2 7.8 8 0 7.9
Table IV. Cystine Content of Hair Reduced with 0.72 >Y Thioglycolate (pH 9.2) and +liylatecl without Previous Drying Cystine Found, Treatment of Hair minutes, alkylated with ethyl bro-
7c
4.5,4.4 3.9,3.8
mide for 18 hours Reduced 8 minutes, alkylated with iodoacetic acid 15 minutes in boiling water bath
3.8,3.9 3.0.3.2
V O L U M E 19, NO. 6
406 Table V.
Effect of Time on Degree of Reduction of Human Hair
[Commercial cold waving solution (DCR-3) a t room temperature] Reduction Time Cystine Found Average Man. % % 17.0,16.0 0, control 16.5 4 8 14
3.0, 2.6, 2.4, 2.5,
20
Table VI.
2.9 2.6 2.6 2.4
3.0 2.6 2.5 2.5
Effect of Time on Degree of Reoxidation of Reduced Human Hair
Oxidation Time Min. 0, control 3 6 9
Cystine Found
Average
%
%
15
on reduced hair was studied. In Table VI the results of a typical experiment are tabulated. The hair was reduced a t room temperature for 15 minutes by the immersion technique using a commercial cold waving solution. Samples of the hair were then transferred t o a 3y0 aqueous solution of potassium bromate, corresponding to a commercial cold waving oxidizing solution. They were left in the solution for varying lengths of time as indicated in Table VI, and then “alkylated” by the iodoacetate method, and the amount of residual cystine was estimated by the Sullivan method.
If one takes into consideration that a 15-minute reduction period brings the residual cystine value down to 37, or less, this experiment shows that most reoxidation takes place in the first 3 minutes. Other oxidizing agents like hydrogen peroxide act much the same as bromate. ACKNOWLEDGMENT
rlus cysteine value of the original hair, and subtracting the value or the residual cystine of the modified hair yields the amount of cysteine formed in the reduction step. Application of this method of analysis to the process of cold waving has yielded some interesting results. I n Table V the effects of reducing a sample of hair by immersion for varying lengths of time in a commercial cold waving solution (0.72 N in thioglycolate and pH 9.2) are listed. The results obtained show that by the immersion technique the reduction is practically complete within 4 minutes and that further treatment has little effect upon the residual cystine. However, this holds true only with solutions such as were used in this experiment. Stronger and more alkaline solutions will disintegrate the hair completely. I n another series of experiments the effect of oxidizing agents
The authors wish to take this opportunity to thank Milton Harris for his interest in this work and for his many helpful suggestions. LITERATURE CITED
(1) Brdzcka, Czechoslov. Chem. Commun., 5,238 (1933). (2) Folin and Looney, J . Biol. Chem., 51,421 (1922). (3) Hessand Sullivan,Ibid., 151,635 (1943). (4) Miller and Du Vigneaud, Ibid., 118,101 (1937). (5) Mirsky and Anson, J. Gen. Physiol., 18,307 (1935). (6) Okuda, J . Biochem. Japan, 5,201 (1925). (7) Patterson, Geiger, Mirell, and Harris, J . Research National Bur. Standards, 27, 89 (1941); Research Paper RP 1405. (8) Sullivan, U.S. Pub. Health Service, Pub. Health Repts., 41,1030 (1926); 44,1421(1929). (9) Sullivan, Hess, and Howard, J . Biol. Chem., 145,621 (1942). PREEENTEDbefore the Division of Biological Chemistry a t the 110th Meeting of the AMERICAN CHEMICAL SOCIETY, Chicago, 111.
Measuring the Volumetric Expansion of Solid Materials W. W. PENDLETON AND H. M. PHILOFSKY, Westinghouse Research Laboratories, East Pittsburgh, Pa.
A method for measuring the coefficient of cubical expansion of solid materials is described. Similar in principle to the A.S.T.M. test for the volumetric expansion of bituminous materials, it presents modifications in both apparatus and procedure which allow use of laboratory equipment and shorten time of testing. Both expansion and contraction may be measured with this method.
T
HIS paper describes a simple method for measuring the volumetric expansion of materials which are in the solid state a t some temperature above the freezing point of mercury ( -38.9 ’ (2.). The method is applicable to measuring the coefficient of expansion of a substance over the temperature range -35” to 300” C. (-31” to 572“ F.). Thermoplastic materials, as well as crystalline solids, may be measured. In principle, the method proposed is the same as that described by Abraham ( I ) , which is the standardized A.S.T.M. test for measuring the coefficient of expansion of bituminous compounds (2). In the standard test, a special steel cylinder, closed a t one end and threaded a t the other, is used for the sample cell. A steel cap is screwed onto the cell and gasketed against a shoulder on the cylinder. Into the cover is welded a steel capillary with such a construction that, when the sample and mercury fill the cylinder and the cap is in place, enough excess mercury is extruded through the capillary to ensure void-free conditions within the cell. During the heating, which is carried out in a liquid bath on the inverted cell, the escaping mercury is collected and carefully weighed. Account is taken of the mercury expansion and steel expansion in the calculation of the expansion of the specimen.
The material may be previously degasified by vacuum and heat in a separate container or in the steel cell with a second cover fitted with a vacuum outlet. Since weighing is the most accurate method of determining volume change, this method should result in very good accuracy. The modifications to the standard test, which are described in this paper, permit the use of ordinary laboratory equipment, shorten the time of sample preparation, and make possible a study of contraction of a material as well as expansion. The method may be used t o measure both “true” and “apparent” volume expansion. As explained by Abraham ( I ) , engineers may wish to know the expansion of a material either as received or after special pretreatment. If the sample is not pretreated, and contains foreign inclusions such as water and dissolved gases, these inclusions will produce an apparent expansion greater than the true expansion. Thermoplastic materials can be degasified t o give true expansions but, with solid materials having no melting points below disintegration temperatures, only apparent expansions may be measured. Although this method has been used only for measuring the volumetric expansion of asphalts (which are thermoplastic), it is applicable to any solid material
