Hence the boil-up rate may be kept constant by automatic control of the pressure drop through the column. For this application, the instrument varies the heat input, or alternatively reduces the operating pressure as required to maintain a constant pressure gradient through the fractionator. A wiring diagram for control by heat input or by pressure reduction is shown in Figure 10. The instrument is normally connected to regulate the heat input to the kettle. For this purpose the following items of equipment are used: a differential mercury manometer equipped with an electrical contact in each leg, an electronic relay, a mercury-plunger relay, and a variable resistor. The differential pressure manometer is inclined; one electrical conductor enters through a packing gland and is movable. Fritted-glass plates (coarse grade) are placed between the kettle and mercury reservoir and between the column condenser and the manometer leg. The fritted glass allows gases t o pass and prevents mercury from leaving the manometer. The electronic relay is employed to achieve a very low current and voltage across the manometer contacts, thus avoiding the possibility of igniting hydrocarbon vapor in the system. The current is about 3 pa. a t 6.3 volts, alternating current. .4n enclosed mercury-plunger relay load switch is used t o eliminate open arcing in the instrument case. A variable resistor is wired in series with the kettle heater element and is automatically cut into and out of the circuit as the electrical contact in the differential manometer is closed and opened. The autotransformer (Powerstat) is manually set a t a voltage which is more than adequate to maintain the desired load in the column, and the variable re-istor is manually set so that when it is in the circuit, the heat input is not adequate to maintain the desired load. To reduce surges within the
fractionator, the high and low heat inputs should be adjusted for a relatively small differential. The alternative method of controlling the column differential pressure is by regulating the rate of reduction in column pressure. This method is used during the period of change-orer from one standard pressure to another. The same instrument components are used, except that the electronic relay actuates a solenoid valve which is placed b e h e e n the column and vacuum source, instead of switching a resistor into the heater circuit. The kettle heater is usually switched off during this period. A three-pole, double-throw switch (Figure 10) permits selection of either of the two methods of control. This semiautomatic control of distillation rate saves man-hours and column time. Bring-up time is reduced and down time required for pressure reduction is eliminated. Furthermore, the quality of data is improved by maintaining a constant boil-up rate throughout the distillation. SAFETY FEATURES
Vent System. All gases vented from columns and vacuum pumps are introduced into a 3-inch metal duct and exhausted from the laboratory. An auxiliary exhaust fan is placed in the outside wall of the laboratory t o remove vapors Tvhich may accumulate in the room. Fire Protection System. Conventional fire protection equipment, consisting of hand fire extinguishers and fire blankets, is readily available. Metal pans are placed beneath kettles to confine spilled materials. I n addition, the laboratory is protected with a permanently installed carbon dioxide system. A valve manifold is arranged so carbon dioxide can be directed to blanket simultaneously all of the stalls on any one of three sides of the room. Nozzle outlets are located
in each kettle zone as well as behind each bank of stalls. The valve manifold and carbon dioxide cylinders for the fire protection system are placed in a hallway near the entrance of the laboratory. EXPERIMENTAL
The new column room arrangement has been in operation for about 5 years. At the present time perforated glass plate, metal packed, and rotary ribbon columns are being used. With this variety of columns, boil-up rates varying from about 100 ml. per hour to 4 gallons per hour may be obtained. Repeatability varies, depending on column types, operating pressures, reflux ratios, and volumes charged. Examples of the repeatability obtained for a perforated glass plate column (30plate, 1 inch in inside diameter) in the fractionation of a hydrocarbon charge are shown in Table I. The reflux ratio was 4 to 1 and five determinations of each volume were made. These values are estimated to be a t least 25y0 better than those obtained by manual techniques. Electrical maintenance has increased, owing to the various automatic innovations introduced. However, this increase is offset by the reduced amount of time required in making column type changes, since less breakage is experienced and little or no manifold variation is required. LITERATURE CITED
(1) Collins, F. C., Lantz, \-ernon, IND. ENG.CHEM., A N A L . ED. 18, 673
(1946). ( 2 ) Willingham, C. B., Taylor, W. J., Pignocco, J. M., Rossini, F. D., J . Research h-atl. Bur. Standards 35, 219 (1945). RECEIVEDfor review May 17, 1956. Accepted March 12, 1957. Division of Analytical Chemistry, 129th Aleeting, ACS, Dallas, Tex., April 1956.
Determination of Tyrosine and Tryptophan in Proteins W . L. BENCZE and K A R L SCHMID Department of Medicine, Harvard Medical School, and Massachusetts General Hospital, Boston 7 4,Mass.
b A new spectrophotometric method for the determination of tyrosine and tryptophan in proteins is based upon measuring the absorbance in the range between 278 and 293 mp. The slope of the line drawn tangent to the two characteristic maxima of the absorption curve is indicative of the content and ratio of these two amino acids. The error introduced in these determinations b y the influence of the
bathochromic shift in the absorption spectra of tyrosine and tryptophan is diminished as compared with that of earlier methods. This method was further applied to the determination in certain proteins of the hydrogenbonded nonionizable tyrosine.
S
determination of tyrosine and tryptophan in proteins (1) offers an advantage over chemiPECTROPHOTOMETRIC
cal methods because it does not require hydrolysis, which often leads to partial decomposition of these two amino acids (6, 11, IS,16,16). The contentof tyrosine and tryptophan established by the spectrophotometric methods of Holiday (7, 8 ) and Goodwin and Morton ( 5 ) is calculated from absorbances measured a t two definite wave lengths. A large error is introduced in these results by the hathochromic shift in the absorption VOL. 2 9 , NO. 8, A U G U S T 1957
1193
spectra of tyrosine and tryptophan (1). The spectrophotometric method presented here represents a n attempt to overcome some of the difficulties inherent in the earlier methods, which are based on the equation of Vierort (20). The new method mas developed on the basis of two distinct features of the absorption curves: First, the slope of the line drawn tangent to the two characteristic maxima indicates the ratio of tyrosine to tryptophan. Second, the extinction at the higher maximum is a measure of the tyrosine-tryptophan content. Both the slope of this tangent as well as the maximal absorption are direct functions of the ratio and amount of these two amino acids. I
I
PROCEDURE
Mixtures of Tyrosine and Tryptophan. The method for determining tyrosine and tryptophan in proteins was based on t h e results obtained by investigating the ultraviolet absorption curves of mixtures of these two amino acids in their free form. Commercial amino acids \\ere recrystallized from a mixture of ethyl alcohol and water, and dried in vacuo a t room temperature over phosphorus pentoxide. Working solutions of these amino acids were prepared to contain tyrosine or tryptophan in known concentrations between0.08 and0.12 m M by diluting aqueous stock solutions with 0 . 1 S sodium hydroxide. Extinction Coefficients of TyrosineTryptophan Mixtures. All measurements were carried out in a Beckman Model DU spectrophotometer or a Cary automatic recording spectrophotometer with l-cnl. cells. The wave length setting of t h e instruments was checked carefully. The absorbances of the amino acid working solutions described above agreed with t h e extinction coefficients reported in the literature ( 1 ) . The values of represent the maximal absorption, in the range between 280 and 293 mp, of solutions of which the sum of the two amino acids mas 1%. The extinction coefficients varied from 134 for tyrosine alone to 266 for tryptophan alone, depending on the molar ratio of the mixtures (Table I). Determination of Molar Ratio and Tyrosine-Tryptophan Content. The absorbances of mixtures of tyrosine and tryptophan were measured between 278 and 294 mp at 2-nip intervals and also a t 320, 330, and 360 nip. The readings were plotted against wave length. The two maxima appeared a t about 282 and 292 mp as a result of tryptophan and ionized tyrosine, respectively. A line m s drawn tangent to these two characteristic peaks (Figure 1).
The slope ( a / b ) of this line was determined by dividing the absorbance intersection ( a ) by the wave length intersection ( b ) . The a i b ratio was again divided by the maximal absorbance, A,,,, of the absorption curve.
1 194
ANALYTICAL CHEMISTRY
I
I
I
I
I
300 mp Figure 1. Determination of tyrosine-tryptophan molar ratio from slope of tangent to absorption curve 250
/b) IO3 s=-( aAm,, -
AA.lO3/A mp
Am,,
=-96,'
- 28 -
0.376
- 9.0
R = 0.5; E = 214. Because of the particular angle of the tangent to the absorbance curve prearrited in this figure, the numerical value of b carries a negative sign. For convenience. the values, !!/A,,,,, 6
called S in Table I, were multiplied by lo3. The S values \\-ere correlated with the E and R values a4 indicated in Table I. I n this way the method became independent of the scale used for the absorbance and wave length. The total content of these two amino acids was obtained by dividing the maximal absorbance of the absorption curve by the corresponding E value : Table I. Principal Measurements for Tyrosine-Tryptophan Determination Molar Ratio, Extinc- Reference Tyrosinetion Values, Tryptophan, Coefficient," R E;:% s Trypto266 ... phan 251 0 1 -17.5 0 2 240 -14.7 230 0 3 -12.6 221 -10.7 0.4 214 0 6 - 9.0 0 7 202 - 6.1 192 0 9 - 3.9 189 1 .o - 2.8 1 1 186 - 1.8 1 3 180 0.11 1 6 176 1.5 2 0 169 + 4 8 2 5 164 8.4 3 0 I61 f11.9 4 0 154 $18.4 5 0 150 ...
++ +
6 0 10 0 20 0
148
, . .
142 ... Tyro138 ... Fine .. 134 ... a If E:%,, values were converted t o molar extinction coefficients, the following equation was used: (201 181 R ) Etyr+try = E!%, lO(1 n,
+
+
Titration of Tyrosine-Tryptophan Mixtures to 1 to 1 Ratio. Table I indicates t h a t the E or S values of t h e tyrosine-tryptophan mixtures and t h e ratio of these two amino acids follow a simple relationship. It was evident t h a t a n y such mixture could be brought t o the 1 to 1 iatio by t h e addition of either tyrosine or tryptophan without impairing this relationship. The 1 to 1 ratio was chosen as the end point of such titrations because the change of the slope of the tangent to the tyrosine-tryptophan absorption curves is maximal \\-hen these two amino acids are in or close to this ratio. This principle was applied in checking the ratio of tyrosine to tryptophan i n proteins (Figure 2). First, the ratio was determined from the absorption curve, then titration was carried out ta the 1 to 1 ratio and the original ratia was redetermined by difference. Method. Protein solutions were prepared by dissolving 0.2 t o 0.5 mg. of protein per ml. of 0 . l S sodium hydroxide. These solutions were kept a t room temperature. Most of t h e solutions were investigated immediately. Depending upon the tyrosinetryptophan content, the maximal absorption usually varied from 0.3 to 0.7. After the absorbance of t h e protein solutions mas measured, t h e absorption curves were drawn from which the content and ratio of the tlvo amino acids were calculated as described above. Spies and Chambers (16) have investigated the stability of proteinbound tryptophan in O.L\- sodium hydroxide a t 25" C. These authors concluded that slight destruction of tryptophan occurs on brief standing of
protein solutions in O.IN sodium hydroxide. The amount of destruction depended on the composition of the protein, the maximum observed loss being 13.4%. This point was not investigated in the present work. The method gave accurate results when proteins free of extraneous absorption were investigated. If the absorption of the proteins such as orosomucoid ( 9 , 1 4 , 2 1 )and lysozyme vias very nearly the wme as that of the corresponding mixture of tyrosine and tryptophan, it \\as assumed that the extraneous absorption was negligible. An excellent criterion for the presence of extraneous absorption was the comparison of the absorbance of a protein a t 320 mp n i t h the corresponding value derived from a mixture of free tyrosine and tryptophan in the same ratio and with the same maximal absorbance.
I
250
!I
300
mM Figure 2. Titration of orosomucoid with tryptophan
The method has not yet been worked out for proteins with extraneous absorption. However, if a correction for the extraneous absorption (Aext) was applied, such as indicated by Beavan and Holiday ( I ) , a fair determination of the content and ratio of tyrosine and tryptophan was obtained
EXAMPLES
Orosomucoid. T h e absorbance of a solution containing 7.07 mg. of orosomucoid (corrected for moisture) in 10 ml. of 0.1iY sodium hydroxide at 360, 320, and 290 mp was 0.021, 0.040, and 0.711 (A,,,), respectively. Correction for the extraneous absorption
according to Beavan and Holiday, amounting to 0.053, was not used because the same correction was obtained from a mixture of tyrosine and tryptophan in a ratio of 3 to 1 with the same A,,,. The slope of the tangent to the absorption curve indicated a tyrosine-tryptophan ratio of 2.9 and an E value of 161. The tyrosine-tryptophan content was
0.711 = 4.42 mg. per 100 161
~
ml. of solution or 6.25% of the protein. Assuming a molecular weight of 48,000 and a ratio of 3 to 1, orosomucoid would contain 12 moles of tyrosine and 4 moles of tryptophan. For the titration, amounts of tryptophan corresponding to 6, 7 , S, and 9 moles, respectively, were added to the orosomucoid solution (Figure 2). The tyrosine-tryptophan ratio of 1 to 1 was observed after the quantity equal to 8 inoles of tryptophan had been added. Recalculation of the molar tyrosinetryptophan ratio resulted in the true value of 3 to 1. Consequently, the content of tyrosine was 4.5% and that of tryptophan l.7yo, Independent deterniinations of the tyrosine content by the nitrosonaphthol method (19) and the method of Moore and Stein gave values of 4.25 and 4.65y0, respectively. The molar tyrosine-tryptophan ratio was 3.5 to 1, 6.8 to I, and 4 to 1 \Then calculated by the methods of Goodwin and Morton (j), Holiday ( 7 ) , and Pantlischko ( I $ ) , respectively. Lysozyme. The absorption curve of crystallized egg IThite lysozyme (products from Armour & Co. and Worthington Biochemical Corp.) in 0.1S sodium hydroxide appeared to be free of extraneous absorption as judged b v t h e curve obtained from free tyrosine and tryptophan in the corrmponding ratio. Taking a molecular weight of 11,900, this protein was found t o contain approximately 3.2 moles of tyrosine and 7 moles of tryptophan. The molar tyrosinetryptophan ratio has been reported to be 3 to 8 (18), 3 to 7 (IO),and 3 to 6 (3, 17) as determined by chemical and microbiological methods. Based on spectrophotometrical methods, values of 3 to 6 (4) and 0.6 (1) h a r e been reported. Determination of Ionized and Nonionized Tyrosine in Proteins. The method !vas applied t o ovalbumin in order to measure t h e change in t h e ratio of ionized tyrosine to tryptophan as a function of the p H of the protein solution. Ovalbumin (products of Armour &- Co. and Worthington Biocheniical Corp.) n as dissolved in 0 . 0 1 s sodium hydroxide t o give a Aliquots concentration of 9.047,. of this solution were adjusted to pH 12.0, 12.5, and 13.0, respectively (Beckman pH meter, glass electrode, S o . 290-75), and kept a t room temperature, At p H 12, n h r r e only part of the tyro-
sine residues is dissociated, a molar tyrosine-tryptophan ratio of 1.0 to 1.0 was noted, which did not change as a function of the incubation time (curve I, Figure 3). At p H 12.5 a n initial ratio of 1.6 to 1.0 was found (curve 11, Figure 3), which changed to 2.5 to 1.0 within 2 days. At p H 13, the true ratio of 2.5 to 1.0 was observed immediately and no further change occured (curve 111, Figure 3). The latter value represents the ratio of all tyrosine residues to those of tryptophan in ovalbumin. These results confirm those published by Cramer and Neuberger ( 2 ) . DISCUSSION
I n this study, it 15-as assumed that the absorption of the tyrosine and tryptophan residues of a protein represented the difference between observed and extraneous absorption. It was also assumed that the incorporation of tyrosine and tryptophan into the protein did not change the extinction of these two amino acids (1). Furthermore, for the determination of tyrosine and tryptophan, all tyrosine residues had to be in the ionized state. The optimal sensitivity of the method, expressed in a change of the slope of the
170-
-# 2E W 150-
130-
260
280
my
Figure 3. Ultraviolet absorption spec tra of ovalbumin
I. pH 12 11. pH 12.5 111. pH 13
Higher maxima of the absorption curves are identical Eith those obtained from lyG solution ( E : :&,) of tyrosine and tryptophan with same molar ratio. This method permits displacing the three curves along the ordinate. VOL. 29, NO. 8 , AUGUST 1957
1195
characteristic tangent, was observed when tyrosine and tryptophan occurred in, or were close to, equimolar amounts. The addition of only 3 y of one of these amino acids to 1 ml. of a protein solution which had been titrated previously to the equimolar tyrosine-tryptophan ratio and which then had a maximal absorbance of 0.4, shifted the molar tyrosinetryptophan ratio from 10 to 10 to 11 to 10. However, the addition of only 1 y of one of these t\yo amino acids caused a distinct change in the absorbance. The influence of the bathochromic shift in the absorption spectra of protein-bound tyrosine and tryptophan upon the determination of the content and ratio of these tlvo amino acids could be essentially excluded by calculating these values from the maximal absorption. I n contrast to the constant value of this absorption, the wave length a t which it occurred varied according to the bathochromic shift and the ratio of these two amino acids. The bathochromic shift is reported to vary b e h e e n 1 and 3 mw ( I ) . Consequently, the results obtained by the method of Goodmin and Morton, which employed absorbance measurements a t definite wave lengths, were not accurate, as Beavan and Holiday ( 1 ) have already pointed out.
ACKNOWLEDGMENT
The authors wish to express their thanks to David R. Kominz, National Institutes of Health, Bethesda, Md., for the tyrosine determinations by the nitrosonaphthol method and by the method of Stein and Moore, and to John Gergeley for helpful discussions. LITERATURE CITED
Beavan, G. H., Holiday, E. R., Advances in Protein Chem. 7, 319 (1952).
Cramer, J. L., Xeuberger, A., Biochem. J . 37,302 (1943). Fromageot, C., Garilhe, 111. P. de, Biochim. et Biophys. Acta 4 , 509 (1950).
Fromageot, C., Schnek, G., Ibid., 6, 113 (1952).
Goodwin, T. W.,Morton, R. A,, Biochem. J . 40,628 (1946). Hirs, C. H. W.,Federation Proc. 13, 230 (1954).
Holiday, E. R., Biochem. J . 30, 1795 (1936).
Holiday, E. R., Ogston, A. G., Zbid., 32,1166 (1938).
Hughes, W. L., in “Proteins,” Neurath, H., Bailey, K., eds., Vol. IIB, p. 663, Academic Press, New York, 1954. (10) Lewis, J. C., Snell, N. S.,Hirschmann, D. J., Fraenkel-Conrat, H., J . Biol. Chem. 186,23 (1950). ( 1 1 ) Li, C. H., Chung, D., Ibid., 218, 33 (1956).
(12) Pantlischko, &I.,Kaiser, E., iindres, H . , Biochem. 2. 322, 526 (1952). (13) Sanger, F., Advances in Protein Chenz. 7. 111952). Schmid, K., J . Ana. Cheni. SOC.75,
en i1g.i?,). \----,-
Smith, E. L., Stockell, .4.,J . Biol. Chem. 207,501 (1954). SDies. J. R.. Chambers, D. C., ANAL. (17) Thompson, k.R., Bzochem. J . 60,513 (1955). (18) Tristram, G. R., in “Proteins,”
Seurath, H., Bailey, K., eds., Vol. IA, p. 181, Academic Press, Xew York, 1953. (19) Udenfriend, S., Cooper, J. R., J . Biol. Chem. 196,227 (1952). (20) Vierort, in “Spectrophotometry,” Twyman, F., Allsopp, C. B., eds., Hilger, London, 1934. (21) Winzler, R. J., in “Methods of Biochemical Analyses,” Glick, D., ed., Interscience, Sew York, 1955. RECEIVED for revien February 29, 1956. Accepted March 2, 1957. Division of Biological Chemistry, Symposium on Techniques of Biochemical and Clinical Interest, 128th RiIeeting, ACS, Minneapolis, Minn., September 1955. Publication No. 212, Robert W. Lovett Memorial Laboratories for the Study of Crippling Diseases, Department of hledicine, Harvard RIedical School, and Massachusetts General Hospital, Boston, Mass. Grants in support of these investigations received from the Helen Hay Whitney Foundation, L-ew Yorlr, and Xational Institute of .\rthritis and Metabolic Diseases, National Institutes of Health, U. S. Public Health Service (-1-509).
Rapid, Precise Micro Vapor Pressure Method A.
Y. MOTTLAU
Products Research Division, Esso Research and Engineering Co., P.O. Box 51, linden, N. 1.
,The petroleum industry has long felt the need for a more accurate vapor pressure method than the presently used Reid procedure. An apparatus has been developed which, in comparison with the Reid, gives approximately a fivefold advantage in precision, a threefold advantage in speed, and requires a sample charge The new apparatus of only 1 ml. has been so designed that its results correlate with those from the Reid.
T
HE PETROLEUM INDUSTRY has long used the Reid method ( I ) for the measurement of vapor pressures of volatile, nonviscous petroleum products, except liquefied petroleum gases. By modern standards the method is cumbersome, subject to too many errors, and requires a much larger sample than is necessary for accurate vapor pressure measurement. LeTourneau, Johnson, and Ellis (3) have developed a reduced-scale Reid
1 196
ANALYTICAL CHEMISTRY
apparatus which has the distinct advantage of requiring a much smaller sample. Unfortunately, no improvement in precision was gained by scaling down the standard apparatus. Levin, Morrison, and Reed (4) described a simple microapparatus for measuring vapor pressure. The device can be readily manufactured in the laboratory, requires a sample as small as 1 ml., and gives a good direct correlation with Reid vapor pressure. The apparatus is delicate, however, and requires much handling, characteristics which do not recommend it for routine laboratory use. Furthermore, the precision claimed by the authors is no better than that claimed by the Reid method. The vapor pressure of a liquid can be measured by introducing a sample of the liquid into a n evacuated bulb fitted with means for measuring the pressure in the bulb before and after sample introduction. Santora (6) developed a method in this laboratory
based upon the evacuated bulb principle. TT’hile useful in the application for \vhich it was designed, the method never proved to be reproducible enough to warrant its substitution for the Reid method. Santora’s apparatus did not hold the vapor-to-liquid ratio constant; a n unavoidable cold spot existed in the wall of the vapor space a t a point where the sample was introduced; and the means for introducing the sample was not considered satisfactory for routine use. This paper describes an easily operated, relatively precise vapor pressure measuring device based on the evacuable bulb principle, and incorporating the Harris sample introduction system. This system is the mercury-sealed orifice (manufactured by Precision Instruments Co., Baton Rouge, La., and distributed by Consolidated Electrodynamics Corp., Pasadena, Calif.) popularly used for introducing gas and liquid samples into the inlet systems of mass spectrometers. An important
