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E. A. ABRAMSON, D. H. MOORE AND H. H. GETl'NER
ELECTROPHORETIC AND ULTRACENTRIFUGAL ANALYSIS OF HAY-FEVER-PRODUCING COMPONENT OF RAGWEED POLLEN EXTRACT', * H. A. ABRAMSON, D. H. MOORE, AND H. H. GETTNER The Electrophoresis Laboratory and the Departnent of Physiology, Columbia University, and the Medical Sem'ce of Dr. George Baehr and the Laboratories of the Mount Sinai Hoepital, New York C i t y , New York Received October $8, loll I. INTRODUCTION
In the United States, the pollen of giant ragweed and dwarf ragweed are probably among the most common causes of seasonal hay fever and asthma. In order to produce these clinical conditions, it is not only necessary for the pollen grains to collide with the mucous membranes of the nose, bronchioles, and eyes, but it is also neceasary that the molecules responsible for the symptoms diffuse through the mucous membranes into the vascular tissues beneath, where the reaction of the sensitized (allergic) tissue occuw with the antigen (or allergen) of the ragweed pollen. It becomes obvious that one of the most important factors, then, in the production of seasonal hay fever and asthma produced by ragweed and other pollen is the size, shape, and net electric charge of the active molecules found in pollen extracts. It is the purpose of this communication to present what are apparently the first data available on the electrophoretic analysis, the sedimentation constants, and the diffusion constants of some of the electrophoretically homogeneous allergenic constituents of giant and dwarf ragweed pollen (2). The small size observed for the allergenic molecules accounts for the ease with which they penetrate the mucous membranes and provides a simple basis for the great frequency with which ragweed hay fever and asthma are found in the population. 11. METHODS
Commercially available dried giant ragweed and dwarf ragweed pollen (as well as timothy grass pollen) were defatted in a Sohxlet extraction apparatus with petroleum ether. After extraction, 5 to 20 g. of the defatted pollen were suspended for several hours in 100 cc. of phosphate or acetate buffers of different ionic strengths and at various pH values. At first the solutions were dialyzed, but because of the rapid diffusion of pigmented and, as was later discovered, of unpigmented material through the ordinary collodion and Visking sacs, it was decided to eliminate dialysis and to study the undialyzed solutions directly. The conductances of the buffer and of the solution to be studied were made identical, because the conductance of the pollen extract was initially always higher than that of the original buffered solvent. 1 Presented at the Eighteenth Colloid Symposium, which was held at Cornel1 University, Ithaca, New York, June 19-21, 1941. * Thi6 investigation hss been aided by a grant from the Josiah Macy, Jr., Foundation.
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In making electrophoretic runs, three types of Tiselius (16) cells were used: ( 1 ) the large preparative cell, (2) the separation cell, and (3) a longer cell for
studying electrophoretic resolution. Ths electrophoretic patterns were studied either directly by observation of the schlieren bands, by the use of the Longsworth (8, 9) scanning technic, or by means of the Philpot-Svensson method (10, 15). The ultracentrifuge employed was an air-driven centrifuge modelled after that designed by Beams. Only schlieren boundaries were possible with the optical equipment available. For this reason, minor heterogeneity in the boundaries was not discernible with our ultracentrifugal technic. 111. GENERAL DESCRIPTION O F THE ELECTROPHORETIC PATTERN
A . The unpagmented slow ( U S )fractions
The electrophoretic fractionation of giant and dwarf ragweed pollen extracts has been complicated because of the presence of many pigments and because of the low molecular weights of the material to be fractionated. However, isolation experiments with dialyzed extracts were essentially unsuccessful because too much of the material was lost with our dialysis technic. Undialyzed extracts gave much clearer and more useful electrophoretic patterns. The USG (unpigmented, slow, giant) and USD (unpigmented, slow, dwarf) fractions were present in very high concentration and readily isolated. The pigments were also observed and the fast pigments (FPG and FPD) easily isolated. In figure 1 is the result of an electrophoretic analysis of crude giant ragweed extract obtained on June 4, 1940 in 0.067 M phosphate buffer at pH 6.50. At the end of 10 hr. one can observe a very large quantity of homogeneous unpigmented material (USG) and at least four other pigmented fractions which have mobilities close to one another. These are separated from the USG fraction by the sliding flange plates of the electrophoresis cell. The optical densities in the two parts of the cell are very different. The unpigmented fraction did not offer any hindrance to the transference of light; consequently the photograph is white where the unpigmented fraction is present in purified form at the boundary. The characteristic cross-hatching5 of the pigmented forms are present in the rest of the picture. The USG fraction has a large area relative to the areas of the pigmented fractions present. The four pigments illustrated in figure 1 have not as yet been isolated from one another. However, u e have isolated one fast-moving pigment (FPG) for biological study and chemical analysis. The four pigments illustrated in figure 1 can not readily be separated by the electrophoretic technics at present. In figure 2 is another example (also descending boundary) of a 3-hr. run in 0.02 M sodium acetate a t p H 4.5. At this p H precipitation may interfere with the experiment. I n this case, the minor pigments are also separated. There are apparently six minor pigments present in this picture in addition to the USG fraction, which is between the flange plates of the electrophoresis cell and the rest of the cell itself. Figure 3 is a smooth curve of the USG fraction. The symmetrical nature of the smooth curve is indicative of electrophoretic homogeneity. Reversal showed
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no sharpening, but this test is not simple to interpret when diffusion is large compared with the electric mobility. Although we have reported in a preliminary publication that the mobility of the USG component was 0.05 p per swond in 0.05 M phosphate buffer at pH 7.0, the results of a much larger series of experiments indicated that, at this concentration of salt, the mobility is only of that order. The highest value for the electrical mobility of the USG fraction that we have observed is 0.07 p per second in 0.06 M phosphate buffer; the lowest mobility was judged to be about 0.03 p per second in the same buffer. The lowest electrical mobility for the same fraction was observed in a solution containing 0.15 M sodium chloride a t pH 7.4. In the presence of as much salt as this, the electrical mobility of the US fractions becomes too small to measure with any certainty and the peak of the curves can be taken as the zero point. But, as has been mentioned, at lower salt concentrations the US fraction has apparently a small negative potential.
B. Comparison of giant and dwarf ragweed Both giant and dwarf ragweed pollen are usually administered in the treatment of hay fever. I t is generally believed, however, that the material in the dwarf ragweed extract is a more potent allergen. For this reason, a comparison was made of the electrophoretic patterns of both dwarf and giant ragweed extract under identical conditions. The patterns of both the ascending and descending boundaries obtained with crude giant ragweed extract and crude dwarf ragweed extract were compared in M/6 phosphate buffer at pH 7.4 and found to correspond closely. The mobilities of three of the corresponding pigments in the ascending boundaries (of giant and dwarf) were as follows: 0.44 and 0.49 1.1 per second
0.65 and 0.89 p per second 0.93 and 0.95 1.1 per second In one experiment in \rhich the high value of the electric mobility of the USG component was noted at pH 7.1 in 0.067 M phosphate buffer, the US fraction had an electric mobility of 0.07 1.1 per second, whereas the four minor pigments which migrate close together had electric mobilities at the descending boundaries of 0.50 p per second, 0.55 p per second, 0.60 p per second, and 0.65 p per second, In the presence of 0.15 ill sodium chloride at pH 7.4, all of these pigments are not readily separated because the small differences in the electric mobilities become less. Because of the disagreement of the mobilities in the ascending and descending boundaries, it is best at present not to give quantitative significance to these minor pigments but to wait for a satisfactory solution of the dialysis problem which is in progress.
C . Isohtion o j the fast pzgments (FPG and F P D ) In figure 4 are illustrations of the separation of the fast pigment (FPG) in giant ragweed extract. The experiments were done in 0.02 M phosphate buffer containing 0.15 M sodium chloride at pH 7.5. The thin-lined peaks in figures
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xp i T*If IDIN
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the nose and lungs and eyes. The CG and CD series in figures 5 and 6 correspond to the ultracentrifugation of crude giant and crude dwarf ragweed solutions. Taking the first photograph &s zero time, the subsequent photographs were taken after 1, 2 , 3 , 4 , and 5 hr. and after 1.5, 2,3.5, and 4 hr., respectively. There is a close parallelism between the ultracentrifuge data of both giant and the dwarf ragweed solutions. The sedimentation constants calculated from the smoothed curves would be only slightly less than that found for the electrophoretically purified fractions. Although the curves of USG and USD are not exactly parallel to the curves of CG and CD, respectively, the CG and CD curves are parallel to one another. The slight retardation experienced in the crude solution may be due to the presence of the pigments or other materials which
P i o . 7. The diffusion of the colorless component (Trifidin) of giant ragweed extract DIFFUSION
0
- FPG
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I
0.53
a6
loor FIQ.8. Diffusion of the fast pigment of giant ragweed extract
retard the movement of the boundary. The close correspondence between the sedimentation constants, however, indicates that the patterns found in the crude materials are caused primarily by the US fractions of both giant and dwarf ragweed. On direct observation of the boundary during ultracentrifugation, the pigment above and below the boundaries in the CG and CD series is equal in color intensity. In other words, there is no readily observable sedimentation of the pigment incidental to the ultracentrifugation. This was confirmed by direct observation of the FPG fraction in the ultracentrifuge. After 2 hr. ultracentrifugation at about 150,000 g, no separation w&s observed. DIFFUSION EXPERIMENTS
Data obtained for the diffusion conskants of the USG component (figure7) and FPG component (figure 8) show that the colorlesw fraction and the fast pigment
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diffuse rapidly compared with proteins like serum albumin; the curves also provide evidence that homogeneous systems are being observed. The data thus far obtained a t 2°C. indicate that the diffusion constant for USG is 1.7 =t0.5 X lo-' cm.2 per second. The diffusion constant (preliminary data) for the FPG cm.2 per second. The rapidity of diffusion and the fraction is 1.3 i= 0.5 x slowness of sedimentation of both of these components indicate that the major colorless components and the fast pigment have molecular neights which are low when compared with those of ordinary proteins. Calculations based on our values of the sedimentation constant and diffusion constant result in a molecular weight for the USG component of about 5000. This low value of the molecular weight fits in with the fact that the biologically active material is readily introduced into the skin by electrophoresis (1) and readily permeates the mucous membrane of the nose and other tissues. CHEMICAL R E 4CTIONS
A . Colorless fractkon In table 1 are listed some of the reactions obtained with the USG and USD fractions isolated by electrophoresis. The samples tested usually had between 0.2 to 0.5 mg. of total nitrogen per cubic centimeter. The FPG usually had TABLE 1
Reactions of CSG and CSDfractzons zsolated b y electrophoresis ~
TEST
I
Standing in ice box. . . . . Phosphotungstic acid. . . Millon . . . . . . . . . . . . Biuret . . . . . . . . . . . . . . . .. I Xanthoproteic.. . . . . . 1 Trichloroacetic a c i d . . . . ' Sulfosalicylic acid. . . . . ! Heller test . . . . . . . . . . . .I Heat and acetic a c i d . . . . Molisch.. . . . . . . . . . . ~
.I'
USG
Usually clear Heavy precipitate Positive Positive Deep yellow Slight turbidity Turbidity Slight opalescence Opalescence Positive
form^ '
Precipitate may Heavy precipitate Positive Positive Positive Turbidity Turbidity Slight opalescence Opalescence Positive
1 '
~
I
Light precipitate Posttjve Positive Deep yellow Prccipitate Precipitate Positive Opalescence
about one-fifth of this quantity of nitrogen. The data that we are presenting a t this time differ slightly from the reactions obtained in our preliminary report, in that more of the protein tests are now recorded as positive. The outstanding feature of the tests was a very heavy precipitate obtained with phosphotungstic acid, heavier, in fact, than that obtained with 1:30 dilution of serum. A heavy precipitate was also obtained with the Millon reagent. On the basis of these tests, we believe that, in spite of the fact that our colorless components are of low molecular weight, the molecuie is protein-like in nature. It is of interest to compare the data obtained here with those of Stull, Sherman, and Hampton (13). The fraction which they obtained, called fraction I, was colorless and, like our USG and USD components, gave precipitates with phosphotungstic acid, a reddish precipitate with the Millon reagent, and other
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reactions for proteins. However, since these investigators studied dialyzed extracts, the components of which were precipitated with ammonium sulfate at p H 4.0, and since we used undialyzed extracts and made our fractionation at p H 7.4 in the absence of excess salt, we prefer to discuss Merences in the number of pigments and their reactions for a future publication. We have named the USG component Trifidin (in view of its homogeneity by electrophoresis and in the ultracentrifuge as well aa the fact that it is biologically active (q.v.). The corresponding USD component in dwarf ragweed we have called Artefolin.
B . Fast pigment One fraction containing fast pigment that was free of the colorless component gave only slight turbidity with phosphotungstic acid. Another FPG fraction containing only 0.05 mg. of total nitrogen per cubic centimeter was found to have 20 per cent of its nitrogen precipitated by phosphotungstic acid. A pigment mixture containing 0.08 mg. of nitrogen per cubic centimeter had about 30 per cent of its nitrogen precipitated by phosphotungstic acid. Possibly more nitrogen would be precipitated from more concentrated solutions. In these reactions, most of the pigment remained in the supernatant fluid. The FPG fraction containing 0.05 mg. of nitrogen per cubic centimeter showed slight turbidity with the usual protein precipitants but the color of the fraction and its high dilution make definite statements at this time unsatisfactory, However, it was apparent that the electrophoretic fractionation gives a highly purified pigment solution which contains much more pigment on a nitrogen basis than the crude material. BIOLOGICAL ACTIVITY
A . Production of hay fever and asthma The USG component (Trifidin) was sprayed into one side of the nose of fifteen hay fever cases and seven normal individuals. None of the normals reacted. However, nine out of fifteen cases of hay fever responded with symptoms varying from mild hay fever to severe hay fever and asthma. In fact, in one case, an attack of asthma was initiated which lasted three days. I t is of interest that the side of the nose into which the material was sprayed showed the only reaction or the more severe one.
B . Skin reactivity The USG component (Trifidin) was highly skin-reactive. On treated skmsensitive patients, scratch tests were readily obtained with solutions containing 0.0003 mg. of nitrogen per cubic centimeter. The USD component (Artefolin) showed scratch tests of the same order of magnitude. On intracutaneous testing a positive skin test waa observed in a patient with a solution containing as little as 0.000005 mg. of nitrogen per cubic centimeter. Only 0.02 cc. of this solution was injected. Since at low concentrations of substances of this type a good deal
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of adsorption by the glass containers must occur, the value of the nitrogen content given here is fictitious, being much less than actually reported. Comparison of the skin reactions of USG, USD, and FPG fractions has been begun. A rather large number of intracutaneous tests of the reactions of the USG and FPG fractions were made (on treated patients) with two solutions on the basis of their nitrogen content. The comparison of USG and FPG is valid, of course, only if the molecular weight and nitrogen composition are identical, The USG fraction was occasionally slightly more active than the FPG fraction. However, on the basis of a rather large number of experiments, it seems likely that the USG fraction and the FPG fraction have approximately the same skin reactivity (based on direct testing on pollen-sensitive individuals). If too high a concentration of the pigment is employed for testing, that is, more than is ordinarily used in solutions for routine treatment, the pigmented fraction may be irritating. Immunological studies have been begun in collaboration with Dr. Gregory Shwartzman. Both the USG and the FPG fractions show the phenomenon of passive transfer. Animal sensitization experiments are also under way. The results of these purely immunological investigations will be reported elsewhere. DISCUSSION
The widespread occurrence of sensitization of the mucous membranes of the eyes, nose, and lungs makes it logical to believe, on the basis of these experiments, that the ease of direct sensitization depends partly upon the presence of small molecules. A combination of low molecular weight and high allergenic properties would lead readily to diffusion across the mucous membranes and sen~itization.~It seems likely that further electrophoretic and ultracentrifugal investigations on pollen allergens will provide an insight into the special types of antibody production which are observable in the allergies. The antibodies found in the pollen cases are characterized as sensitizing antibodies in the human being. X o precipitins are formed when the serum of sensitive cases is mixed with its allergen (antigen). It is conceivable that the low molecular weight of the antigen may be in part responsible for the lack of precipitin reaction. The electrophoretic method of analysis gives data which may be different from those obtained by chemical fractionation. We refrain, therefore, from a detailed discussion of the experiments of others (3, 4,5, 6, 7, 11, 12, 13) who employed salting out or similar procedures. As noted in the preceding section, the electrophoretic pattern of timothy grass has shown a striking resemblance to those of ragweed. We are planning to survey the electrophoretic patterns and biological activity of a number of the grasses to see if the nature of the components isolated from the grasses parallel
* This is all the more so on the basis of preliminary experiments with timothy gram extract. The electrophoretic pattern of timothy grass also showed a major colorless slowmoving component (UST), with t4e minor pigments quite similar in pattern t o that observed in the ragweed pollen.
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their common immunological activity and to test our thesis that the low molecular weights of the pollen allergens may share part of the responsibility for the frequency of spontaneously occurring sensitization in human beings. SUMMARY
The development of hay fever and asthma depends upon the rate at which the active allergens of the pollen diffuse across the mucous membranes to produce tissue reactions. To understand the importance of this diffusion process in the development of hay fever and asthma, the active components of pollen extracts have been isolated by electrophoretic analysis. In the extracts of the pollen of both giant and short ragweed at or about pH 7.0 there are negatively charged major colorless components which are highly skin-reactive and which produce hay fever and asthma. The giant component (USG), Trifidin, and the dwarf component (USD), Artefolin, were electrophoretically isolated and ultracentrifuged. From the diffusion and sedimentation constants, the molecular weight of each was calculated to be about 5000. The main sedimenting boundary in the crude pigmented extract had approximately the same sedimentation rate as the purified major colorless component. The low value of the molecular weight fits in with the fact that the biologically active material is readily introduced into the skin by electrophoresis. The chemical reactions of the colorless components correspond to polypeptides of high molecular weight or protein-like substances in solutions containing about 0.2 mg. of nitrogen per cubic centimeter. There are usually four to six minor pigmented boundaries as well as the major colorless component. The fastest moving pigments have been isolated and are biologically active. Preliminary experiments with extracts of timothy grass indicate that there are present at least five pigments and a major negatively charged colorless component which migrates very slowly, corresponding to the colorless component of the ragweed pollen extracts. The authors wish to express thanks to Miss S. Goldsmith for assistance and cooperation. REFEREKCES (1) ABRAMSON, H. A,, 8, 272 (1940).
AND
GORIN,M. H.: Cold Spring Harbor Symposia Quant. Biol.
D . H., AND GETTIER,H . H.: Proc. SOC.Exptl. Biol. Med. (2) ABRAMSON, H . A,, MOORE, 46, 153 (1941), preliminary report. H. A. E . , AND GERMAN, J. L. M . : J . Allergy 6, 335 (3) BENJAMIN, C . E., VAN DISHOECK, (1935). F . A,: Southern Med. J . 20,257 (1927). (4) BERNTON, H . S . , JONES,D . B . , . ~ N DCSONKA, (5) CAULFEILD, A . H. W., COHEN,C., A N D EADIE,G. S.: J . Immunol. 12, 153 (1926). (6) CAULFEILD, A . H . W . , BROWN, M . H . , AND WATERS,E. T.: J . Allergy 7 , 1 (1935). (7) JOHNSON, C . A . , AND RAPPAPORT, B . Z.: J . Infectious Diseases 50,290 (1932). (8) LONGSWORTH, L. G., AND MACINNES, D . A , : Chem. Rev. 24,271 (1939). (9) LONQSWORTH, L . G . , SKEDLOVSKY, T., AND MACINNES, D . A . : J. Exptl. Med. 70, 309 (1939).
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(10) PHILPOT, J . S. L.: Nature 141, 783 (1938). R . A., AND CHOBOT, R . : J . Biol. Chem. 92, 569 (1931). (11) STULL,A., COOKE, R . A , , AND CROBOT, R . : J. Allergy 3,341 (1932). (12) STULL,A , , COOKE, W., AND HAMPTON, S . F.: J. Allergy 12, 117 (1941). (13) STULL,A , , SHERMAN, T . , AND PEDERSEN,K . 0.: The Ultracentrifuge. Oxford University Press, (14) SVEDBERG, London (1940). (15) SVENSSON, H . : KolloidLZ. 87, 180 (1939);90,141 (1940). (16) TISELIUS, A . : Trans. Faraday Soo. 93, 524 (1937).
T H E DENATURATION O F PROTEINS AND ITS APPARENT REVERSAL. 111' HANS NEURATH, GERALD R . COOPER,
AND
JOHN 0. ERICKSON
Departmnt of Biochemistry, Duke University School of Medicine, Durham, North Carolina Received October $8, 1941
A number of investigations, purported to demonstrate protein denaturation to be reversible, have been published in recent years. In view of the fundamental importance of the problem for considerations of protein structure, it appears appropriate to undertake a critical examination of the data at hand, and to attempt to decide whether and to what an extent denaturation can be reversed at all. In consideration of the limited space available for the contributions to this Symposium, the discussion will be confined to aspects of the problem as they have evolved from the inyestigations reported in the preceding papers of this series (18, 19) and from previous work on reversible protein denaturation. In the face of limited experimental evidence, such a discussion cannot be entirely free of speculations. Denaturation may be defined as some intramolmular rearrangement of the protein giving rise to definite changes in chemical, physical, and biological properties (1). This rather loose definition is given a clearer meaning by stating the most important changes which have been recognized to accompany denaturation. These are (1) a loss of biological specificity, (8) a decrease in solubility, (3) a loss of crystallizing ability, (4) an apparent increase in the number of specific groups, such as sulfhydryl, disulfide, or phenolic groups, and ( 5 ) an increase in the relative viscosity of the solution of a protein and a corresponding decrease in its diffusion constant, indicative of an increase in apparent molecular asymmetry. A protein having undergone all these changes may be said to be denatured. I t is probably denatured if it has undergone any one of the last three changes, but it is questionable whether denaturation has occurred if any one of the first two changes alone has taken place. A more precise definition of denaturation, as of any chemical reaction, necessitates not only qualitative Presented at the Eighteenth Colloid Symposium, which waa held at Cornel1 University, Ithaca, New York, June 19-21, 1941.
