INDUSTRIAL AND ENGINEERING CHEMIXTRY
April 15, 1931
149
New Substrates for Use in Detecting Proteolytic Activity' Robert L. Jones FREDERICK STEARNS
AND
CO., DETROIT,
MICHIQAN
c
OLORIMETRIC proBY a modified technic, adsorption systems of egg necessary to set the dye and cedures for establishalbumin and dyes have been prepared which function prevent the later liberation of ing the presence of 8 8 excellent qualitative reagents for determining color by either dilute acid or proteolytic enzymes in variProteolYtic activity. Several albumin-dye reagents dilute alkali in the absence ous fluids have long been in have been prepared, two of which have given good reof proteolytic enzymes. use. The s u b s t r a t e s used suits when tested with commercial pepsin, trypsin, consist of some class of protein and artificial gastric and pancreatic juices. The Study of Dyes with which is incorporated a method of preparation and results of the tests with dye in such a manner that diegg albumin-iodoeosin and egg albumin-basic fuchsin Based on the general proare given. These materials are more easily prepared gestion of the former releases cedure outlined above, 5 dyes than carmine-fibrin or Congo red-fibrin, and being dry were studied. These were thelatter and imparts a color to the system. This release powders, are stable indefinitely under ordinary laborachosen with respect to depth tory conditions. of color is therefore dependent of color, relative acid or alkali upon the presence of some fastness, and minimum degree enzyme which will digest the protein-i. e., the presence of free of re-adsorption by the peptized albumin. All tests were run coloring matter in the system is taken as a positive test for in a water bath a t a temperature of 52' C., using approxiproteolysis. Carmine-fibrin and Congo red-fibrin, which are mately 1 gram of substrate. The amounts of digestive often used in routine laboratory instruction in physiological ferments used were considerably in excess of the minimum chemistry, are familiar examples of such materials which func- requirements. The object in this series of tests was to detertion on the above principle. mine the utility of such egg albumin-dye substrates as Fibrin, a derivative of fibrinogen and possessing in general qualitative tests for proteolytic enzymes, with no attempt the properties of the globulins, is not water-soluble. There- at ascertaining the sensitivity of the method. This phase of fore, the preparation of carmine-fibrin and Congo red-fibrin the subject will be touched upon later, in connection with depends simply upon soaking the fibrin in a dilute acid or comparative results obtained with egg albumin-dye and alkaline solution, respectively, and gives rise to a n adsorp- fibrin-dye substrates. Control tests, tubes with no digestive tion system in which the dye is largely held upon the surface ferment added, were run simultaneously. The following reof the insoluble protein. By using a protein such as egg sultswereobtained. albumin, which can be dispersed in water, protein-dye com(1) EGGALBUMIN-GENTIAN VIOLEvThiS combination, binations which simulate true absorption have been pre- tried with one part of 1:3000 commercial pepsin to appared. These substrates, which have been produced by co- proximately 100 parts of substrate, showed pronounced agulation of the protein in a colloidal system containing the release of color within 30 minutes. The depth of color prodye, function as excellent Preparations for demonstrating duced was not suitable for a colorimetric procedure and there Proteolytic activity Dispersion and subsequent coagula- was considerable re-adsorption of the liberated dye by the tion of the protein give more intimate contact with the dye peptized albumin. The control tubes also showed a conand a firmer combination of the two. The experimental siderable release of color. results indicate that protein-dye combinations thus prepared A ~ ~ GIZEEN-ThiS ~ absorption ~ ~ (2) contain a larger proportion of dye, and that i t is more uni- systemunder conditions identical with those in (1) gave formly released as digestion proceeds. very similar results. Although the amount of re-adsorption was not as pronounced as with gentian violet, the degree of Preparation of Substrates color produced was not sufficient. There was an appreciable Merck egg albumin, scales, and native egg albumin have release of colorin the control tubes, been used in the preparation of these substrates, the general (3) EGG ALsuMIN-IoDoEosIN-This dye was the most method Of preparation being as follows: To a per cent satisfactory of any tried in acid systems. When the ratio of system) Of dried egg Or a to 1:3000 commercial pepsin to substrate was 1 to 100, a deep aqueous of fresh egg the appropriate dye pi& color was apparent in 30 minutes in 0.30 per cent hywas added to a concentration Of Oel per cent* A trace Of drochloric acid solution, This color was permanent for 8 acetic acid was added and the system by heating hours and there was no liberation of dye in the control tubes to goo to 90"c. The cOagulumwhich separated Over this interval, Excellent results were obtained with was filtered off by suction and air-dried on a porous plate for this material in artificial gastric juice 112 hours. This dried material was then reduced to a coarse substrate gave (4) E'?G ALBUMIN-BASIC E'UCHSIN-ThiS powder, and washed by shaking five to six times with either 0.30 per cent hydrochloric acid or 0.25 per cent sodium carbon- very satisfactory results in a 0.25 per cent sodium carbonate. The solution used for this washing was dependent ate solution, using one part of 1:25 trypsin to five parts Of in 35 upon the type of proteolytic ferment for which the material substrate. A deep color was imparted to the was to be used as a testi. e., whether active in a dilute acid minutes and no color was released in the control tubes. The or a dilute alkaline medium. The first drying was apparently Permanence of the color indicated that re-adsorption was very slight, I n 20 cc. of 0.25 per cent sodium carbonate 1 Received November 13, 1930. Presented before the Divisfon of containing 3 CC. of artificial pancreatic juice ( 2 ) , egg albuBiological Chemistry at the 80th Meeting of the American Chemical Society, min-basic fuchsin gave more positive results in a shorter Cincinnati, Ohia, September 8 to 12, 1930. 0
~
ANALYTICAL EDITION
150 Table I-Comparison
Vol. 3, No. 2
of Color Liberation f r o m a n d Digestion of Protein-Dye Substrates by Proteolytic Enzymes ~~~
No.
SUBSTRATEu
ENZYME Start
-Egg albuminiodoeosin Egg albuminiodoeosin Carmine-fibrin
1
2 3
Carmine-fibrin Egg albupin-basic fuchsin Egg albumin-basic fuchsin Congo red-fibrin
4 5 6
7
8 Congo red-fibrin le-gram portions.
1 part per 1000 of 1:8000 commercial pepsin
None 1 part per 1000 of 1:3000 commercial pepsin None 1 part in 8 of 1:25 trypsin None 1 part in 8 of 1:25 trypsin None
NITROGEN IN SUBSTRATEFREEFILTRATE
COLOR
I
1I
30 minutes
60 minutes
Colorless
Reddish pink
Deep pink
Colorless
Colorless Deep pink, opalescent Faint pink
Colorless Deep pink, very opalescent Pronounced pink
Colorless Colorless
Start
30 minutes 60 minutes
Mg./cc. 0.02
Mg./cc. 0.19
Mdcc.
0.04
0.04
0 05
0.03
0.04
0.61 0.09
0.70 0.13
0.27
Colorless
Deep orchid
Intense purple
0.06
0.10
0.16
Colorless
Colorless
0.03
0.03
0.05
Colorless Colorless
Deep pink, very opalescent Slight opalescence
Colorless Light red, very opalescent Opalescent, faint pink
0.07 0.03
0.69 0.06
0.96 0.07
8
interval of time than in 1 per cent commercial trypsin solutions. (5) EGGALBUMIN-GENTIAN VIoLET-Results with this material in dilute alkaline solutions containing commercial trypsin were unsatisfactory. A pronounced opacity developed in the tubes containing the proteolytic enzyme, but there was little depth of color, indicating a rapid re-adsorption of the dye by the peptized albumin. (6) EGQALBUMIN-METHYLENE BLUE-This substrate, under conditions identical with ( 5 ) , behaved very similarly, and the results secured were unsatisfactory for the same reasons. Of the 5 egg albumin-dye substrates investigated, two, egg albumin-iodoeosin and egg albumin-basic fuchsin, were found to give very pronounced colorimetric tests for enzymes of the peptic and tryptic type, respectively. Accordingly, these two reagents were compared with carminefibrin and Congo red-fibrin as qualitative tests for pepsin and trypsin. Comparisons were made of the degree of color release from these protein-dye substrates and the degree of digestion as indicated by the amount of nitrogen in the substrate-free filtrate from each test. All tests, together with controls, were run simultaneously in test tubes immersed in a water bath a t 52" C. The results are given in Table I. Tubes number 2, 4, 6, and 8 were the controls, containing no digestive enzyme. Experimental Procedure
Egg albumin-iodoeosin and egg albumin--basic fuchsin were prepared according to the method previously given. The fibrin-dye substrates were prepared according to the usual method (S), using Difco purified, desiccated blood fibrin. Since fibrin preparations are kept under glycerol, whereas the albumin substrates were prepared dry, it was necessary, in order to obtain comparable amounts of each, to soak them for hour previous to use in either 0.30 per cent hydrochloric acid or 0.25 per cent sodium carbonate. The substrates were then filtered on a Buchner funnel and 1-gram portions weighed out for each test. Since the protein-dye materials were weighed moist, the proportion of enzyme to substrate, as stated in Table I, is not accurate. All figures are on the same relative basis, however, and are none the less comparable. To these portions of substrate, 20 cc. of 0.30 per cent hydrochloric acid or 0.25 per cent sodium carbonate containing the correct amount of pepsin or trypsin, respectively, was added. I n the determination of nitrogen 2-cc. portions of the liquid above the substrate were withdrawn, filtered immediately, and nitrogen determined therein by a modification of the Koch-McMeekin (4) method for nitrogen in blood. These nitrogen determinations are given for substrate-free filtrate. With both fibrin preparations a pronounced opacity,
due to finely divided protein particles, developed within 30 minutes. At least a portion of this colloidal material passed through the filter paper. Therefore the determinations were not made on protein-free filtrate. The peptized fibrin in the 2 cc. of filtrate, though still possessing protein characteristics, is in a sense an index of degree of digestion of the substrate. The nitrogen figures throughout may not be comparable, since a certain portion of undigested protein nitrogen is represented in the filtrates from the fibrin substrates, whereas the increase in nitrogen in the filtrates from the albumin substrates is probably due to actual digestion. It appears that liberation of color from fibrin-dye combinations is due to peptizing of the fibrin, the finely divided particles carrying the color and imparting a decided opacity to the system. With egg albumin-dye combinations, no opacity develops and the liquid above the substrate takes on a considerable depth of color. This release of c,olor in the latter case appears to be dependent upon proteolysis, since in the controls (numbers 2 and 6 of Table I) there was no release of color and no increase in nitrogen content of the substrate-free filtrate over a period of 1 hour. With respect to the sensitivity of these modified substrates no definite conclusions can be drawn, since only two amounts of pepsin and trypsin were used. I n the first attempted comparative test of egg albumin-iodoeosin with carminefibrin and egg albumin-basic fuchsin with Congo red-fibrin, one part of 1:3000 pepsin to 3000 parts of substrate, and one part of 1:25 trypsin to 25 parts of substrate were used. The color developed in 60 minutes with any substrate was barely perceptible, certainly not sufficient for a qualitative test. When three times these amounts of ferments were used, the results given in Table I were secured. It appears probable, therefore, that under the conditions of these experiments sensitivity of the fibrin-dye and albumin-dye substrates is not materially different and lies somewhere between the two levels run-i. e., well under the maximum range of peptic and tryptic activity. Conclusions
Although re-adsorption of the dye by the peptized protein is a factor of considerable magnitude in some protein-dye adsorption systems, the resuIts herein presented show that it is possible to select dyes in which this factor operates a t B minimum. These results also demonstrate the possibility of preparing protein-dye combinations which are fast to acid or alkali under the conditions required for the optimum activity of peptic and tryptic enzymes, and in which the release of color is dependent solely upon proteolysis. I n the air-dried condition in which these materials are prepared, it has not been found necessary to use special care or methods in their preservation. Samples which have
INDUSTRIAL A N D ENGINEERING CHEMISTRY
April 15, 1931
been kept in stoppered reagent bottles under ordinary laboratory conditions have shown no evidence of decomposition or loss of reactivity over a period of several months. I n addition to their keeping qualities under ordinary laboratory conditions, egg albumin-iodoeosin and egg albuminbasic fuchsin fulfill the necessary conditions with respect to minimum degree of re-adsorption and acid or alkali fastness. These advantages, together with their relative ease of preparation, demonstrate their possibilities in qualitative tests
151
for proteolytic activity and lead to the belief that such substrates will be found more satisfactory than carmine-fibrin and Congo red-fibrin. Literature Cited (1) Hawk, P. B., and Bergeim, Olaf, “Practical Physiological Chemistry,” p. 210, Blakiston, 1926. (2) Hawk, P. B., and Bergeim, Olaf, Ibid., p. 259, (3) Hawk, p. B., and Bergeim, Olaf,I b i d , , p. 56. (4) Koch, F. c., and McMeekin, T. L., J . A m . Chem. s ~ c 46,2068 ., (1924).
Absolute Determination of Nitrogen in Organic Compounds’ Pregl’s Micro-Method Otto R. Trautz and Joseph B. Niederl DEPARTMENT OF CHEMISTRY, NPW YORK UNIVERSITY,
F ALL the methods in
NEW
YORK, N. Y .
The accuracy of Pregl’s micro-Dumas method may to keep the degree of purity of organic microanalysis be increased by using measured amounts of carbon the carbon dioxide constant. the absolute nitrogen dioxide and Copper oxide, and aPPlYing empirical corThe Kipp of Figure 1 can be rections for air and absorption errors. Wall adhesion determination enjoys perhaps evacuated and can then dethe greatest use and the field (0.5 Per cent of the nitrogen volume collected) and liver carbon dioxide containof its application in organic vapor pressure of the Potassium hydroxide solution ing onlv 0.001 cc. imDuritv in are considered. and bioidgical as well as ih in1 6 cc”. c a r b o n dioxfde. dustrial research is steadily Other satisfactory arrangeincreasing. Quite a number of investigations have been deal- ments are described by, Niederl, Trautz, and Saschek (4), ing with the various phases of the method in order to explain and by Lindner (S), Diepolder (I), and Hein ( 2 ) . The comand eliminate sources of error. The following comprehen- bustion tube and the azotometer are filled according to disive description of the analytical apparatus and procedure rections given by Pregl(5). may be of assistance, therefore, to those availing themselves Method of this reliable and quick method for the determination of nitrogen. A sample of 3 to 5 mg. is weighed on a standard microApparatus chemical balance (sensitivity 0.001 mg.), or on an ordinary The analysis train, as shown in Figure 1, consists of a analytical balance (sensitivity 0.1 mg.) making use of the Kipp carbon dioxide generator, gasometer, combustion tube, dilution method (4). For the introduction of the sample into CT,two-way stopcock, E, and precision azotometer. The the combustion tube Pregl’s (6) instructions are followed, care acid of the Kipp must be guarded from contact with air in order being taken that for each analysis the same amounts of the coarse and especially of the fine copper oxide are used. 1 Received November 20, Then the air in the combustion tube must be eliminated by 1930. Presented under the title passing one gasometer charge of carbon dioxide through the “Improvements in the Microsystem while cold (stopcock E open to the atmosphere), Dumas Method” before the Division of Physical and Inorganic If near the end of this operation, which should require about Chemistry at the 79th Meeting 5 minutes, stopcock E is opened to the azotometer, small of the American Chemical Somicro-bubbles (not larger than 0.2 of one division) should ciety, Atlanta, Ga., April 8 to appear in the azotometer. 12, 1930.
0
Figure 1-Diagram
of Apparatus
