HOW TO D E T E R M I N E STRUCTURAL FORMULAS FOR PROTEINS1* WILDER D. BANCROFT
AND
HOWARD F. BROWNING
Department of Chemistry, Cornell University, Ithaca, New York Received August 1, 1997
The biochemists are agreed that the determination of the structural formulas of proteins is a very important problem. They are also agreed that they do not see any promising method of attacking the problem. The matter does not seem to us to be quite so serious as that, for we think that we know how it should be attacked. With proteins having apparent molecular weights of 15,000 and upwards, the ordinary methods of fractional hydrolysis, isolation, and purification are futile. An entirely new technique must be devised. Fortunately, that new technique is available. The equivalent weight of gelatin with reference to the stoichiometric addition of hydrogen chloride gas is about 300 (2, 4). We may therefore consider gelatin as made up of statistical groups or units, with an average equivalent weight of 300 with reference to the stoichiometric addition of hydrogen chloride to nitrogen, or with reference to stoichiometric nitrogen, as we shall call it. These units are only statistically of the same size and will necessarily vary in composition, because it is not possible to put radicals of all the amino acids to be obtained from gelatin into one unit and keep the equivalent weight as obtained down to 300. The problem is therefore to devise as many ways as possible of breaking up the protein molecule, so as to throw as much light as possible on the composition of the actual units. It is not likely that the protein molecules are absolutely unsymmetrical, and the action of enzymes shows a tendency to break into groups vhich are not identical for the different enzymes. For the moment it is necessary to make an exhaustive study of the types of nitrogen in any given protein and of the JTay in which these relative amounts vary with progressive decomposition and rearrangement of the protein. I n any protein or any mixture of hydrolyzates or other decomposition products of that protein, it is possible to determine total nitrogen 1 Presented a t the Fourteenth Colloid Symposium, held a t Minneapolis, Minnesota, June 10-12, 1937. 2 This work is part of the program carried out a t Cornell University under a grant from the Heckscher Foundation for the Advancement of Research, established by August Heckscher a t Cornell University.
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WILDER D. BASCROFT AND HOWARD F. BROWNING
(Kjeldahl), so-called amino nitrogen (T-an Slyke), so-called stoichiometric nitrogen (Bancroft (l)),and free ammonia. It is possible also to determine how much formaldehyde can be added and, within limits, how it will affect the types of nitrogen already specified. Levy (8) reports that only the amino groups of arginine, histidine, and lysine are considered as reacting with formaldehyde, each of them reacting with one or two molecules of fornialdehyde successiyely.” It is probable that other characterizations of nitrogen will be found when people concentrate on this point ( 5 ) . Gurin and Clarke (6) report that “the position of the free amino groups in polypeptides and proteins can be determined by benzenesulphonylation, followed by hydrolysis under conditions in which the peptide linkages are opened without appreciable attack on the sulphonamino grouping. . . . Treatment of gelatine with benzenesulfochloride blocks the free amino groups completely with no apparent degradation of the protein. , . , At least fifty per cent of the free amino groups in gelatine may therefore be ascribed to the €-amino group of lysine. Xot more than five-tenths per cent of the free amino nitrogen in gelatin can be allocated to monoamino acids.” We advise doing fractional hydrolysis of a protein, say gelatin, Ti-ith water, acid, alkali, pepsin, trypsin, and erepsin; also fractional photochemical decomposition, taking the word “photochemical” in its broadest sense, to include cathode rays, canal rays, emanations and other bombardments. At the end of the short run, the system should be separated into two (or more) portions varying in composition, by filtering off any insoluble portion, by fractional solution, by fractional precipitation with alcohol, acetone, or any other suitable agent, by electrodialysis, etc. The two (or more) portions are to be evaporated to dryness in the cold and examined without purification for the different types of nitrogen. Other runs can be made with the original substance for longer times and any fraction may be treated further. It is worth noting that the proteoses are soluble in benzaldehyde (3), while the amino acids and the proteins are not, egg albumin being something of an exception. In general, isolation and purification of individual compounds will be carried out only at such stages as seem desirable, thereby saving enormous amounts of time. Some of this time will be lost, owing to the slowness of the present method of determining stoichiometric nitrogen, but it is probable that methods of speeding up these determinations mill be found. Czarnetzky claims t o have made some improvement, but he gives no figures and his data for ammonia are known to be wrong. The value given in his thesis for the dissociation pressure of hydrated sodium sulfate is badly in error, and the value in the journal article is even more incorrect. It does not appear definitely XThether his experiments were done ((
. STRUCTCRAL FORMULAS O F PROTEINS
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a t constant partial pressure and varying total quantity of ivater or a t constant total quantity and wrying partial pressure of water, presumably the first. Both ways are wrong unless suitable corrections are made. As these corrections were not mentioned, they were probably omitted. Czarnetzky has also overlooked the fact that a flat in a concentrationpressure curve indicates only the appearance of a new phase of approximately constant coiiiposition and does not necessarily mean the forniatioii of an aiiimoniuni salt. It is quite possible, however, that ammonia might combine stoichiometrically with some of the hydrolysiq products of casein, in which case such experiments might be helpful in our problem. \Then a niass of data has been obtained under suitably controlled conditions, it will undoubtedly be a terrible job to fit the jig-saw puzzle together, but a t least we shall have the pieces, vihereas now we face the adiiiittedly impossible problem of constructing the puzzle from nonexistent pieces. We feel certain that the differences between the enzyme hydrolyses and the pH hydrolyses will be marked and will prove very helpful. Our own experiments were cut short by the illness of hIr. Browning in 1932 and will probably not be carried to a definite conclusion because of the retirement of the senior author. In 1932 we had not realized the iiiiportaiice of doing fractional precipitation on the soluble hydrolyzates, and consequently the only definite results are that the water-insoluble or noli-peptizable portion acts in some respects like unchanged casein, and that the soluble or peptizable hydrolysis products change in composition while there is still what behaves in some respects like unchanged casein. This means that some of the more hydrolyzed products of casein are hydrolyzed somewhat more rapidly than some of the less hydrolyzed products and makes it not impossible that some catalyst Tvould cut out pretty completely one of the intermediate products, just as nitrite can be made to occur only in small amounts in the reductioii of nitrate. After experiments with both concentrated and dilute hydrochloric and sulfuric acids, dilute sulfuric acid (4.44 per cent) was decided to be the best for several reasons: (a) The use of hydrochloric acid involves the determination of chlorides, which is complicated by the presence of so much organic matter. ( b ) Hydrolysis with hydrochloric acid gives a small, presumably variable, amount of black humin substance (lo), while sulfuric acid gives only a trace of it. (c) The soluble portion froni the hydrolysis with both concentrated (20 per cent) and dilute (6 per cent) hydrochloric acid is extremely hygroscopic after being dried and powdered, so that it is difficult to weigh out samples, while the product obtained after hydrolysis with sulfuric acid is much less hygroscopic. ( d ) Concentrated acids produce such rapid hydrolysis that the first stages cannot be observed, while this is not the case with the more dilute acid. (e)
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WILDER D . BANCROFT AND HOWARD F. BROWNING
Besides not producing so much black “humin” substance, sulfuric acid can be neutralized by an equivalent weight of barium hydroxide, and the resulting precipitate of barium sulfate can be removed by filtration. The Kjeldahl method for the determination of total nitrogen (7) and the method of T’an Slyke (11) for the determination of amino nitrogen were used throughout. For the determination of the amino nitrogen in the insoluble portions of the short-time hydrolyzates, a very fine suspension in water of the weighed sample was made up in a volumetric flask and an aliquot portion of this suspension was used. The Van Slyke apparatus was shaken by motor for five minutes for all determinations. For the work with 4.44 per cent sulfuric acid, exactly 100 cc. of the acid was added to 10 g. of the ~ a s e i n . ~ When it was desired to stop the hydrolysis, the flame was removed and the mixture was filtered ininiediately with suction on two thicknesses of filter paper. The residue on the paper \vas washed with several portions of hot water, and the washings were combined with the first filtrate. There was an insoluble portion after the l-hour hydrolysis and after the 2-hour hydrolysis; but not after the 4-hour hydrolysis. The insoluble portion was dried in a desiccator over sulfuric acid. The total and amino nitrogens and the curve with hydrogen chloride were determined on the dried material after it had been powdered finely. The soluble portion from the hydrolysis was treated by heating the first filtrate quickly to boiling, adding an amount of powdered barium hydroxide equivalent in weight to the sulfuric acid used, and mixing this thoroughly with the solution by shaking. The mixture was again heated quickly to boiling and the barium sulfate was filtered off with suction on two thicknesses of filter paper and washed twice with hot water. The washings n-ere combined with the filtrate, which was then evaporated nearly to dryness in an oven held at 65-75OC. When the product had dried to a very viscous mass, the dish was placed in a vacuum desiccator which ivas then exhausted. After standing several days the product could be powdered easily. The original casein contained 14.8 per cent nitrogen and 1 g. of it combined stoichiometrically with about 90 mg. of hydrogen chloride. This means that 23.3 per cent of the total nitrogen is what we call stoichiometric nitrogen. The statistical group or unit is therefore about 416, as against about 300 for gelatin. During the hydrolysis with 4.44 per cent sulfuric acid, ammonia was set free, practically all of it in the first hour of hydrolSince the casein had 3 Casein. refined, prepared b y Schering-Kahlbaum, Berlin. an ash content of only 1 2 per cent, n-e considered i t unnecessary t o purify it further for the preliminary orienting n o r k t o be described in this paper. Rancroft and Barnett (J. Phys. Chem. 34, 479 (1830)) have shown t h a t a small amount of impurity does not change the curve for stoichiometric nitrogen much.
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STRUCTURAL FOR3fULAS O F PROTEIXS
ysis. The apparent losses in grams of ammonia per 100 grams of casein were: 1 hour, 3.84 g.; 2 hours, 3.83 g.; 16 hours, 3.83 g.; 32 hours, 3.80 g. The data are shown graphically in figure 1. The average of 3.85 g. of ammonia correspoiids to 3.16 g. of nitrogen or 21.3 per cent of the total nitrogen. This value is adopted in table 1. These results do not agree quantitatively with those of Pittom (9) obtained over twenty years ago. He found that the rate of ljberatioii of ammonia was very rapid in the first stages of the hydrolysis and that, after that, the ammonia comes off a t a
Hours
FIG.1. Loss of nitrogen as ammonia Hydrolysis
TABLE 1 casein with 4 44 per cent sulfuric acid Variation of the soluble portion 0.f
h 1 A T E R I l L AS-D T R E A T X E S T
i
STOICHIOMETRIC SITROGEN
,
APPRoXIMATE LOSS O F
NOK-STOICHIOMETRIC NITROGES
rlTRoGEr AMhlOS1.A
I
per cent
Original c a s e i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-hr. hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-hr. hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . 4-hr. hydrolysis.. . . . . . . . . . . . . . . . . . . . . . . . . . 32-hr. hydrolysis.. . . . . . . . . . . . . . . . . . . . . . . . . .
23.3 34.5 37.5 ,
44.1
53.1
per cent '
i
1
715.7 44.2 41.2 34.3 25.6
, ~
~
'
per cent
0 21.3 21.3 21.3 21.3
steady, slow rate for a time. The great discrepancy is that Pittom obtained only about 1.5 per cent of the total nitrogen in the form of ammonia, whereas we get about 3.15 per cent, or practically twice as much. Pittom's coiiditioiis of hydrolysis were quite different from t'hose in our experiment, and that may possibly account for the difference in the results. The matter should be looked into, because it would be very interesting and probably helpful, if the liberation of aninionia could be varied considerably by varying the conditions of hydrolysis. T H E JOCIlICAL OF P H l S I C A L C H E J I I S T R Y , V O L .
41,
NO.
9
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WILDER D. BANCROFT A S D HOWARD F. BROWXIKG
The insoluble portion of the casein, left after 1 and 2 hours' hydrolysis with 4.44 per cent sulfuric acid, showed the same percentage of stoichiometric nitrogen as the original casein within the limits of the experimental error and was, therefore, probably unchanged casein. The data for the stoichionietric addition of hydrogen chloride are presented graphically in figure 2. Curves -1and B are for unchanged casein; curve C is for casein after 1-hour hydrolysis; and curve D is for casein after 2-hour hydrolysis. I n order to differentiate these curves it was necessary to enlarge the scale of absciww. This niakes the actual break very blurred. Figure 3 s h o m what can be done by a judicious choice of scale. Dr. R. A. Gortner does not believe that any casein could survive 2 hours' boiling with dilute sulfuric acid and he therefore considers the iiisoluble product as a decomposition product. It is true that only one kind of test was applied, and it is possible that casein may deconipose
Mill grams HCI per Gyam Casein
FIG.2
/Siliqrarns
n'Gl per
Gram Hydro/ys/s iZodocr
FIG 3
FIG.2. D a t a for stoichiometric addition of hydrogen chloride. Curves A and B, unchanged casein; curve C, 1-hour hydrolysis; curve D , 2-hour hydrolysis. FIG.3. Curves repiesent I-hour, 2-hour, 3-hour, 16-hour, and 32-hour hydrolysis.
in such a way as to keep the percentage of stoichiometric and of total nitrogen constant, but it does not seem probable. One point, which supports Gortner's view, is that the 3-hour hydrolyqis did not give appreciably more animoiiia than the 1-hour hydrolysib and one would have expected an additional amount, coining from the casein assumed not to have been decomposed in the firft hour. This difficulty would be met if it were shown that the presence of hydrolysis products of casein interfered with the setting-free of ammonia. This has not yet been shown, but is an additional reason why someone should study carefully the conditions under which ammonia is split off. Some of the results that rvere obtained with the hydrolyzed portions are summarized in table 1. The stoichiometric nitrogen, as found, increases from 23.3 per cent of the total nitrogen to 53.1 per cent in the product which has been hydrolyzed
STRUCTURAL FORMULAS O F PROTEIKS
1169
for 32 hours. If we include the ammonia nitrogen as stoichiometric nitrogeii-~vhich of course should be done-the change is from 23.3 per cent to 74.4 per cent and gives some indication of the probable helpfulness of studying stoichionietric nitrogen. At the end of 32 hours’ hydrolysis the amount of hydrogen chloride combining stoichiometrically with 1 g. of hydrolyzed casein (exclusive of ammonia) had risen from about 90 mg. to about 265 nig. The data are shown graphically in figure 3. If one is going to undertake seriously the determination of the constitution of any protein, he must be willing to accept facts as they are, and that is not the case at present. Since the living organism can hydrolyze proteins more or less completely t o amino acids and can synthesize the needed proteins from amino acids, and since the hydrolytic decomposition can also be done in the test-tube, it has seemed self-evident to everybody that the peptide linkage -C(: 0)YH- must predominate in the proteins. We even speak of certain decomposition products of proteins as polypeptides, without having as yet any proof that these substances are polypeptides. It has been shown by Bancroft and Barnett that the nitrogen in a peptide linkage is normally stoichiometric nitrogen. In casein not over 20 per cent of the nitrogen linkages can possibly be normal peptide linkages and the number may be considerably less than that. Zein contains apparently no stoichiometric nitrogen and therefore no normal peptide linkages. That conclusion must be either accepted or explained away. COSCLUSIONS
1. Sulfuric acid is better than hydrochloric acid for hydrolyzing casein and is probably better for hydrolyzing all proteins. 2. When casein is hydrolyzed for a short time, there is apparently some unchanged casein left. Dr. R. A. Gortner does not accept this conclusion, and we are not in a position to prove that he is wrong. The soluble hydrolysis products vary in coniposition while there is still present what we call unchanged casein. 3. Under the conditions prevailing in our experiments practically all the aninionia is split off during the first hour of hydrolysis. The percentage of total nitrogen split off as ammonia is about 21.3. 4. The stoichiometric nitrogen increases froni 23.3 per cent of the total nitrogen in the original casein to 53.1 per cent in 32 hours’ hydrolysis if one does not include the ammonia nitrogen, and to 74.4 per cent if one does. 5 . One gram of the original casein combines stoichiometrically with about 90 mg. of hydrogen chloride. This makes the equivalent weight with respect to stoichiometric nitrogen about 415. After 32 hours’ hydrolysis the value (exclusive of ammonia) has dropped to about 140. 6. I t is claimed that great progress in the determination of constitu-
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WILDER D. BANCROFT AXD HOWARD F. BROWSING
tional formulas of proteins will be made if we discard temporarily the idea that only pure substances should be analyzed. 7. It is believed that it will be helpful t o study the amounts of rarious types of nitrogen in suitably prepared, fractional hydrolyzates of the proteins. 8. The importance of stoichiometric nitrogen must be recognized, and also the corollary that the normal peptide linkage is not so prevalent as organic chemists have assumed. 9. The important problem is to devise methods for cleaying the protein molecule into relatively large, natural fractions rather than to nibble off small fragments from the circumference of the molecule. That is why the enzyme attack will probably be most helpful. REFERESCES
(1) BANCROFT ASD BARSETT:J. Phys. Chem. 34, 449, 753, 1217, 1930, 2433 (1930). (2) BELDES:J. Phys. Chem. 36, 2164 (1931). (3) COOPERAND ASHLEY: Biochem. J. 19, 533 (1925). (4) CZARNETZKY AND SCHMIDT: J. Biol. Chem. 106,301 (1934). (5) EAGLE AND VICKERS:J. Biol. Chem. 114, 193 (1936). (6) GURIN. ~ N DCLARKE:J. Biol. Chem. 107, 395 (1934). (7) KJELDAHL: Z . anal. Chem. 22, 366 (1883). (8) LEVY:J. Biol. Chem. 109,365 (1933). (9) PITTOY: Biocheni. J. 8, I57 (1914). (IO) PLIVMER: The Chemical Constitution of the Proteins, Vol. I, p . 65 (1917). (11) VAN SLYKE:J. Biol. Chem. 10, 13 (1912).
