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Just before the mercury reaches stopcock Sz, it is closed, and evacuation is completed in the remainder of the system. No157 oxygen is slowly admitted to the system through SS until atis opened and the mospheric pressure is reached, after which ,'% pressure in the nitrometer tube is allowed to rise to atmospheric by additional oxygen admitted through 83. To msure the removal of all air from the system, this procedure is repeated two additional times. In the following manner the final adjustment of pressure is made: In the assembly of the apparatus, mercury was added to B1 until its level in the nitrometer tube was a few millimeters above the first graduation. At this point in the adjustment, a pressure, slightly above atmospheric, is placed on the system by the manipulation of SS,thereby depressing the mercury level in the nitrometer tube. Now, 8 1 is opened to the electrolysis cell, B4,which is at atmospheric pressure, and then 84 is closed, isolating the gas in Bz from the system, In this procedure the final step is the adjustment of pressure in the system to bring the level of mercury in U-tube H up to contact 1. This is done by allowing gas to leak from the system, through SI, until the pressure in Bzis sufficiently above that in B3to complete the circuit of contacts 1 and 2. The flash of light, caused by the ionization of the Grid-Glow tube in the electronic relay, instantly shows when mercury reaches contact 1. As a result, the pressure in Bz is about 1 mm. above atmospheric, but the pressure in B3 and in the reaction flask is approximately atmospheric. The automatic control of pressure, in the apparatus just described, is based on the pressure of the gas in BZ when & is closed. When a slight amount of oxygen is used up by the sample, the pressure in Ba is decreased by a corresponding amount. Consequently, this drop in pressure allows the gas in Bzto force the mercury in H upwards, until contact 1 is closed. When this happens, the grid in the electronic relay loses control,
and the tube passes current through the electrolysis cell, B4, The gases thus formed pass through 81into BI, causing an increase in pressure and a rise in the mercury level in the nitrometer tube. This decreases the volume of the system until the original pressure, which breaks contact 1 and stops the electrolySiS, is restored. To operate the device, only a small fraction of a millimeter difference in pressure between Bz and B3is required. In a normal run, bulb Bz serves a further purpose when the temperature of the bath is rising continually. An increase in temperature tends to cause an increase in volume of the gas in the system; but, since the tendency is equal in Bz and B3,and since Bz is closed from the system, the two tendencies are counterbalanced through U-tube H . The gas in the system is, therefore, of constant specific volume, and consequently, no correction factors are applied to the readings of the nitrometer tube. Figure 4 shows temperature increase and volume absorption curves, plotted as a function of time, for a typical run. At 50" C. these curves deviate only slightly from straight lines, but do show an upward tendency in 20 hours. For a given sample of bituminous coal, the correlation between temperature increase and oxygen absorption is sufficiently good t o predict one accurately from the other.
Literature Cited ( 1 ) Davis, J. D., and Byrne, J. F., J.Am. Ceramic Soc., 7, 809 (1924) ; IND.ENG.CHEM.,17,126 (1926). (2) Howard, H. C., Ibid., 13,231 (1921). (3) Kohman, G. T., J.Phys. Chem., 33,226 (1929).
RECEIVED April
16, 1936.
Location of the Antienzyme in Egg White J . S. HUGHES, H. 31. SCOTT,
AND
J. ANTELYES, Kansas Agricultural Experiment Station, Manhattan, Kan.
F
OR many years i t has been known that raw egg white resists proteolytic digestion. At first i t was thought t h a t this resistance was due to the inherent character of the various proteins of the egg white. Sugimoto, according to Needham (S), was the first to show that egg white contained a n antienzyme which was responsible for this resistance to proteolytic action. Balls and Swenson ( I ) , while studying proteolysis in stored eggs, found that this antienzyme was located in the thin white. The thick white was found to contain a sufficient amount of trypsionogen to liquefy i t a few hours after activation with enterokinase. I n this work Balls and Swenson did not separate the thin white into the inner and outer fractions. Since these two kinds of thin white are formed by entirely different processes (9) and serve different functions, there is no reason to assume t h a t they are alike in antienzymatic action.
Method In this investigation the method described by Balls and Swenson was followed, with the exception that the thin white was divided into inner and outer portions. The portion of the egg white to be tested was incubated for 30 minutes with a solution of enterokinase, buffered with an
ammonia-ammonium chloride mixture, and a solution of casein was added. The acidity of this mixture was determined immediately by titrating an aliquot portion with 0.1 N alkali, using thymolphthalein as an indicator, The remainder of the mixture was incubated 30 minutes, after which the increase in acidity was taken as a measure of the proteolytic activity. The egg white was divided into three fractions-outer thin, thick, and inner thin-by breaking the egg into a Petri dish. A sample of the outer thin was removed with a pipet, and the remainder of the outer thin was carefullly removed with filter
paper. The thick portion was then cut with scissors to allow the inner thin to run out. A sample of this inner thin fraction was pipetted off and the remainder of this fraction was removed with filter paper. The remaining thick white was separated from the yolk and chalazae with a suitable pipet. The thick white was finally forced through a fine-meshed screen to render it soluble in the digestion mixture. While this procedure is not suitable for quantitative determination of the amount of each fraction, i t does give a representative sample of each of the three fractions for analysis. Samples from five eggs were pooled for each determination which was made in the afternoon on eggs laid t h e same morning. I n addition to determining the proteolytic activity of the three fractions separately, determinations were also made on mixtures of equal volumes of inner thin a n d thick, and outer thin and thick. The results are given i n Table I.
TABLE I. PROTEOLYTIC ACTIVITYOF EGGWHITE (Diffenenaes i n acidity for the various fractions and mixtures, before and after incubation, as measured in cc. of 0.1 N alkali.) Thick Thick DetermiOuter Inner and Outer and Inner nation Thin Thick Thin Thin Thin 0.95 0.18 0.16 0.65 0.52 -0.30 -0.44 0.06 0.10 0.04 -0.04 0.08
0.1s
0.13 -0.14 0.11
0.60 0.54 0.90 0.50 0.24 0.49
0.01 -0.26 0.01 -0.13 -0.07 -0.10
0.64 0.64 1.22 0.52 0.06 0.56
0.02 -0.16 0.43 0.09 -0.08 0.02
I n each of the seven determinations the thick white showed distinct proteolytic activity, which is in accord with the results of Balls and Swenson. When the inner thin was mixed with the thick in equal proportions, the proteolytic activity
JULY 15, 1936
ANALYTICAL EDITION
was reduced in each case. I n contrast with this, when the outer thin was mixed with the thick, the proteolytic activity of the mixture was greater than t h a t of the thick alone in five cases, practically equal in one, and less in one of the seven determinations. These results indicate t h a t most of the inhibitory substance responsible for the resistance of raw egg white of freshly laid eggs to proteolytic activity is located in the inner thin fraction.
311
Literature Cited Balls and Swenson, ENa. CHEM,, 26, 570-2 (1934). (2) Hughes, Scott, and Antelyes, unpublished data. (3) Needham, "Chemical Embryology," Vol. 111,p. 1307, New York, Macmillan Co., 1931. RECEIVEDApril 27, 1936. Presented before the Division of Biological Chemistry at the 9 l s t Meeting of the American Chemical Society, Kansas City, Mo., April 13 t o 17, 1936. Contribution No. 204, Department of Chemistry, Contribution No. 96, Department of Poultry Husbandry.
Preparation of Flattened Copper Tubing Coils EDW.IRD P. BARRETT AND WILLIAM L. BARRETT, Bureau of Mines, Pittsburgh, Pa.
numerous joints made by this method has failed; they are tight and strong, and do not decrease the cross-sectional area of the flattened tubing. The details of the joint arc shown in Figure 2. A is the jig for forming the sleeve, B the parts of the joint, and C the completed joint in a section of flattened copper tubing. A piece of copper tubing about 1.25 inches long, having an inside diameter equivalent to the outside diameter of the tubing to be joined, was shaped on the jig, A , the cross section of which was the same as that of the flattened tubing to be joined. If a piece of the larger tubing is not available, the short piece may be easily made by turning and drilling a piece of copper rod.
FIGURE1. FLATTENED COPPERTUBING COILIN
THE
Two methods of completing the joint have been used: (1) The inside of the sleeve and about an inch of the end of each piece of tubing were coated with silver solder. Borax was used as a flux. The silver-solder-coated ends were inserted in the sleeve as shown in C and sweated together by heating with a smalltipped oxy-acetylene torch, after which the ends of b were silver-soldered t o a and c,
e+-
MAKING
F
LATTENED copper tubing may be used more advantageously than circular tubing in many types of laboratory and experimental apparatus. FORMING THE FLATTENED TUBING.Flattened copper tubing may be readily formed by drawing circular tubing through special dies. I n 1926 William L. Barrett made two dies for forming flattened copper tubing: one for forming tubing 0.5625 X 0.1875 inch with an opening 0.4375 X 0.0625 inch from 0.375-inch copper tubing having 0.0625-inch wall; the other for forming tubing 0.75 X 0.21875 inch with an opening 0.625 X 0.09375 inch from 0.5625-inch tubing having 0.0625-inch wall. MAKINGCOILS OF FLATTENED TUBING. Numerous spirally wound coils of flattened copper tubing have been made in the metallurgical laboratories of the Pittsburgh Station of the United States Bureau of Mines, a mandrel being made for each diameter of coil required. The photograph, Figure 1, shows a coil in the making. The mandrel, M , 7 inches in diameter, is mounted in a lathe. Flattened copper tubing N was formed as circular copper tubing C was drawn through the die, P,and simultaneously wound on the mandrel. The mandrel was hand-rotated by means of bars inserted into the holes, R. The curved tool, 8, held the turns together and prevented the flattened tubing from twisting as it was wound onto the mandrel.
JOINING FLATTENED COPPERTUBING.It was often necessary to join pieces of flattened copper tubing. None of the
' FIGURE
\LY 2.
METHOD OF
I
Y
JOININGFLATTENED COPPERTUBING
respectively. (2) The ends of the tubing were inserted in the sleeve as shown in C and the ends of b silver-soldered to a and c, respectively. RECEIVBD April 27, 1936.
