574
CATHERINE F. C. MACPHERSON AND MICHAEL HEIDELBERGER
tion through an intramolecular reaction analogous to the well-known intermolecular Michael reaction. Unlike the latter, however, the intramolecular reaction is not inhibited by alkyl substituents on the a- and P-carbon atoms of the acceptor group, and it appears to go substantially -
Vol. 67
to completion. The products are derivatives of 2-carboxycoumaran-3-acetic acid. The reaction was devised and is being further studied as a possible route to synthetic compounds related to morphine. MINNEAPOLIS, MINNESOTA RECEIVED DECEMBER 29,1944
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[CONTRIBUTION FROM THE DEPARTMENTS OF BIOCHEMISTRY AND MEDICINE, COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA ~JNIVERSITY,AND THE PRESBYTERIAN HOSPITAL, NEWYORK CITY]
Denatured Egg Albumin.' I. The Preparation and Purification of Crystalline Egg Albumin Denatured in Various Ways2 BY CATHERINE F. C. MACPHERSON AND MICHAEL HEIDELBERGER
Nowhere is the need for more precise information on denatured proteins more urgently implied than in the all-inclusive definition of denaturation given in the most recent review of the subject.a The prevailing utter lack of agreement as to the criteria of denaturation would seem to render a descriptive approach to the problem permissible, the more so as the original object of the present studies, the use of acid-denatured egg albumin (DnEa) as an intermediate, was thwarted by the extreme difficulty of obtaining successive preparations with reproducible properties. These dit% culties, in part induced by the desire to avoid, as far as possible, degradation' of the egg albumin (Ea) used, necessitated a critical study of the preparation of acid-DnEa, and to this was added a similar investigation of other methods for the denaturation of Ea and the comparison and .attempted correlation of the chemical, physical, and immunological properties of the products. The criterion of denaturation employed was the insolubility, a t the isoelectric point, of the product, DnEa, derived from the isoelectrically soluble native protein, hen egg albumin. Qualitative immunological studies by Wu, Ten Broeck and Li6 on egg albumin (Ea) denatured by acid, alkali, heat, and alcohol showed the denaturation products to be closely related to each other except that only alkali-DnEa did not react with antiserum to Ea and Ea did not react with antiserum to alkali-DnEa. Flosdorf and Chambers6 found that denaturation by intense sound (1) A summary of this series of papers was read before a joint meeting of the New York and New Jersey Sections of the Society of American Bacteriologists on M a y 16, 1942, at Princeton, New Jersey. (2) The work reported in this communication was carried out in part under the Harkness Research Fund of the Presbyterian Hospital and has been submitted by Catherine F. C. MacPherson in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University. Original manuscript received July 6, 1944. (3) H. Neurath, J. P. Greenstein, F. W. Putnam and J. 0 . Erickson, Chem. Rcv., 84, 157 (1944). (4) Degradation is here defined as the splitting off of nitrogenous or sulfur-containing portions of the labile protein molecule. (5) H. Wu, C. Ten Broeck. and E. P. Li, Chinese J . P h y s d . , 1, 277 (1927). (6) E W. Flosdorf and L. A . Chambers. J. ImmumI., 48, 297 (1935).
vibrations produced the same qualitative immunological changes as heating with acid or alkali. Mirsky7,demonstrated that the same number of sulfhydryl groups was liberated in Ea denatured by urea, guanidine, Duponol P. C., heat, or shaking. Each of these comparative studies dealt with only one property of DnEa and little attention was paid to the state of aggregation8 or degradation4 of the products, factors which not only would be expected to influence the chemical, physical, and immunological properties of DnEa but which also, if neglected, would cause confusion as to the changes attributable to denaturation alone. While it might be inferred from the few ultracentrifugal studiesg on the particle weights of various kinds of DnEa that the molecules of these substances are associated or aggregated in water containing only just enough alkali to dissolve them, little, if anything, is known of the degree of association brought about by a particular denaturant or how this might be affected by variations in either the concentration of denaturant or the reaction time or temperature. Experimental Ea was prepared by the method of Kekwick and Cannan.1° Four-times recrystallized Ea was air-dried and dialyzed against distilled water in the presence of toluene at 0-5' until no more sulfate was removed. Toward the end of these studies it was found that by allowing the saltfree isoelectric Ea solutions of varying opalescence to stand at 37" until all precipitate settled, any DnEa contained in them was removed, leaving a water-clear supernatant. 1. Acid Denaturation of Ea.-The denaturation of Ea by acid at a concentration of about 2.4 rng. Ea N per ml. was carried out by addition of 0.1 M HCl to bring the pH to 1.5 or 2.0. The final normality of the acid was usually about 0.05 or 0.025, respectively. The pH, determined with the glass electrode, remained constant during the reaction. In most instances the course of the de(7) A. E. Musky, J . Gcn. Physiol., a l , 725 (1941). ( 8 ) Aggregation is defined as the linking of DnEa molecules, assuming M. W. DnEa = M. W. Ea = ca. 40,000. into aggregates or polymers. Opalescence and gel-formation were considered visual evidences of the process, useful for guidance during the preparative work. More substantial evidence is given in the text. (9) (a) A. Rothen, Annals N. Y. Acad. Sci., IS, 229 (1942); (b) J . B. Nichols, Tars JOURNAL, 61, 5176 (1930). (10)R. A. Kekwick and R. K,Cannon. Biockrm. J . #SO,282 (1828).
April, 1945
PREPARATION OF DENATURED CRYSTALLINE EGGALBUMIN
575
naturation was studied quantitatively at constant tem- twenty minutes 1.5-ml. samples of the reaction mixture were withdrawn, precipitated with trichloroacetic acid, perature. Varying volumes of reaction mixture were pipetted o”,c filtered; and compared in matched test-tubes with the and added to 3 ml. of 0.1 M acetate buffer at PH 5 at 0 . same volume of Ea standard filtrate after simultaneous Because 2 to 3 ml. of reaction mixture were needed for addition of gum ghatti solution and Nessler reagent to estimation of DnEa during the early stages, it was neces- both. The time was noted at which ammonia could first be detected, and a larger preparative run was then allowed sary to add a few drops of 0.1 N alkali to the buffer-protein solution in order to ensure isoelectric precipitation. to proceed for one hour less than this time. In spite of All precipitates were washed twice in the cold with 3-ml. this, other degradative changes were not avoided since a portions of buffer as already described.” In the process of faint odor of hydrogen sulfide could be detected as soon washing precipitates of DnEa form films on the surface. as the reaction mixtures were neutralized. These break into small fragments on mixing, and are partly Salt-free reaction mixtures containing alkali-DnEa were lost when the supernatants are decanted, interfering with always water-clear, so that aggregation in excess of normal the accuracy of the analyses. The difficulty may be incurred during purification was readily detected by the largely avoided by addition of saponin, up to 0.05%, to appearance of opalescence. the buffer used for washing.’* The validity of the method 3. Denaturation of Ea in Other Ways.-Conditions was established by preliminary tests which showed quanti- used for the denaturation of Ea by heat or by trichlorotative recovery of acid-DnEa from mixtures of solutions of acetic acid are given in Table 11. The insoluble precipitate Ea and DnEa in different proportions. (H10-DnEa) which separated from a dialyzed, isoelectric The rate studies permitted no clear-cut conclusions as .solution of Ea during the course of 1 yr. is also included for to the order of the reaction. Approximately straight lines comparison. were obtained up to at least 50% conversion when either 4. Purification of the Denatured Roteins.-Jn the log [Ea] or l / [ E a ] was plotted against time. There was following general purification procedure conditions causing also considerable variation in the rates at which various increased dpalescence and gel-formation are avoided preparations of Ea were denatured, as noted in Table I. by carrying out all operations a t 0” in low salt concentraSimilar variation was observed recently for the action of tion and by redissolving the material promptly after each urea on tobacco mosaic virus.Ia centrifugation. When the reaction has progressed as far as desired, the TABLE I chilled reaction mixture is diluted with cold distilled water COMPARISON OF RATESOF DENATURATION OF VARIOUS so that the concentration of sodium chloride will not exceed 0.01 M after neutralization. The PH is adjusted to PREPARATIONS OF Ea BY ACID the isoelectric range by rapid addition of about 90% of Extent the calculated amount of 0.1 M acid or alkali, followed of denaby the remainder in small portions. After each addition PH Reaction turaEa at which Concn. of acid or alkali, an aliquot of the mixture is centrifuged. DnEa prepn. deNaC1, Temp., time, tion, When the supernatant of the test sample is water-clear, hr. % M OC. natured prepn. used the main lot is centrifuged at 0°.14 The precipitate is 46 50 1.96 0 25 17 7 then homogenized with a volume of distilled water equal 21 50 2.01 0 25 22 8 to the volume of the mixture at the initial precipitation, and the minimum amount of 0.1 M alkali necessary to 26 6.8 50 7 1.51 0 16 dissolve the protein is added with swirling. Reprecipita25 5.5 50 8 1.53 0 21 tion and re-solution are repeated as described until samples 11.5 50 0 25 9 1.53 of two successive supernatants remain clear when boiled 22d 60 0.1 R.T.’ 1“ 1.50 or do not react with anti-Ea rabbit serum. The N concentrations of the fourth and fifth superna3“ 1.50 0.1 R.T. 22d 94 tants of the products usually ranged from 0 to 0.003 mg. 24.5d 26 0.5 R.T. lb 1.96 per ml. Since one or two additional reprecipitations 24.5d 44 0.5 R.T. 33 1.96 yielded Supernatants in the same concentration range, Simultaneous runs. b Simultaneous runs. R. T. = purification was often discontinued when the level of 0.003 mg. N per ml. was reached. The product was finally disroom temperature. Approximate. solved at PH 7.5 a t an approximate concentration of 1% Reaction mixtures of acid-DnEa were always opalescent, and stored in the ice-box with toluene as preseflative. but the opalescence disappeared during aging after the This is referred to as the “aging” process. In no instance products were isolated under the ,conditions recommended was there reason to suspect bacterial contamination. The below. Such preparations were considered “normal.” only soluble material appearing in any supernatant after Lots of acid-DnEa showing unusual opalescence, or in- aging and subsequent isoelectric precipitations was Ea (see creasing opalescence during less cautious manipulation below). All supernatants from alkali-DnEa became turbid when warmed to room temperature. Occasionally were listed as “aggregated.” concen- supernatants from aged acid-DnEa behaved similarly. 2. Alkali Denaturation of ’Ea.-Approximate trations of the reactants for all preparations were 2.4 mg. The small amounts of precipitate which settled over a Ea N per ml. and 0.04 N NaOH. The stock 0.10 N period of two daoys did not dissolve when the supernatant NaOH used contained less than 0.005 M BaCII. Since the was cooled to 0 . No such turbidity was observed when secondary hydrolytic effect of alkali was evident early in all operations were carried out at room temperature. the course of denaturation, attempts were made to avoid Therefore, nitrogen analyses on alkali-DnEa supernatants degradation by determining the time at which analytically were run on aliquots allowed to warm to room temperature detectable amounts of free ammonia first appeared. Fif- and filtered only after precipitation was complete. The teen ml. portions of Ea-alkali mixture were placed in a precipitate collected from the supernatants of alkali-DnEa thermostat a t 25“. A sample of the original Ea solution, 23, Table 11, was purified by isoelectric precipitation and diluted to equal N content, was precipitated with one- found to show the same quantitative immunological bethird vol. of freshly prepared 20yotrichloroacetic acid and havior with antiserum to alkali-DnEa as did DnEa 23. Acid-DnEa which was not allowed to remain in the isothe filtrate used as a standard. At intervals of fifteen to electric state for more than approximately sixteen hours (11) (a) M. Heidelberger and F. E. Kendall, J . E x g f l . Mcd.. 66, during purification and was not in contact with more than 555 (1932): (b) M. Heidelberger, F. E. Kendall and C. M. SooHoo, 0.02 M salt (a “normal” product) usually exhibited a ibid., 68, 137 (1933). bluish opalescence during the first week of aging and was (12) S. D. Kenriksen and M. Heidelberger, J . E x p f l . M i d . , T4, 105 (1941). (13) G. L. Miller, 1.Bwl. Chwn.. 146,888 (1942).
(14) Precipitates of alkali-DnEa packed better on centrifugation than did those of acid- or heat-DnEa.
CATHERINE F.c. MACPHERSON AND
576
HEIDRLBERCER
MICH.4EL
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TABLEI1 PREPARATION, PURIFICATION, DEGRADATION AND BEHAVIOR ON AGINGOF DnEa Preparation ---Denaturation-
7
--EaWt. Product Lot N, T$mp., DnEa,No. no. mg. gH C. Acid We 7 1011 1.51 26 25 Add17 7 644 1.96 Acid 21 8 201 1.53 25 Acid22 8 201 2.01 25 Acid32 9 125 1.55 R. t.' R.t. Acid33 9 160 1 . 4 8 .. . ... Acid 33B Alkali23 8 180'0.041N' ' 25 Alkali28 8 413 .041Nbsk 25
..
.
Alkali 30 Alkali24' Alkali 18"'
8 7
597 404 561
Heat25" Heat 29O Heat 31
8 8 9
154 6.8* 165 6.Sb 83 7 . 3
6
.040Nc*' .040Nk .048Nk
25 25
25 76 80 100
Time, hr. 49 107 49 108 29 37
...
2 1.5
1.1 3.7 43
Ex-
tent,
%
90.5 74.5 97 89 ..
.. ..
