1117
VARIATIOS I N PROTEIN CONCENTRATION IS PLASMh
(15) LANGMUIR, I.: Cold Spring Harbor Symposia Quant. Biol. 6,171 (1938). (16) MARBLAND, D. A , : In The Structure of Protoplasm, edited by W. Seifriz. Iowa State College Press, Ames, Iowa (1942). (17) NORRIS,C. H.: J. Cellular Cornp. Physiol. 14, 117 (1939). (18) KORRIS,C. H.: J. Cellular Comp. Physiol. 16, 313 (1940). (19) KORTHEN,H . T.:Botan. Gaz. 100, 238 (1938). (20) NORTHEN, H. T., AND KORTHEN, R. T. Plant Physiol. 14,539 (1939). R . W.,AND STREET,S. F: Biol. Symposium 3,9 (1941). (21) RADISEY, (22) SEIFRIZ,W.: Am. Katuralist 80, 121 (1926). (23) SEIFRIZ, W.: Am. Naturalist 63, 410 (1929). (24) SEIFRIZ,W.:Protoplasm. McGraw-Hill Book Company, Inc., New York (1936). (25) SEIFRIZ,W : The Structure of Protoplasm. Iowa State College Press, Arnes, Iowa (1942). (26) SICHEL,F.J. M.: J. Cellular Cornp. Physiol. 6,21 (1934). (27) WAUGH,D.F.,AND SCHJIITT,F. 0.: Cold Spring Harbor Symposia Quant. Biol. 8, 233 (1940).
.
NORM.4L VARIATIOK I S T H E CONCENTRATION OF FIBRINOGEN, ALBUMIN, AND GLOBULIN IN BLOOD PLASMA’ ROBERT hl. HILL
AND
VIRGIXIA TREVORROW
Department of Biochemistry and the Child Research Council, University of Colorado School of Medicine, Denver, Colorado Received August 4 , 194.9
Ten years ago, impressed by the lack of adequate data in the literature, we began a study of the concentrations of the albumins, globulins, and fibrinogen in the plasma of healthy individuals and the changes that these fractions undergo with age. The protein fractions were separated by a micro-modification of the “Howe technique,” Le., the fibrinogen and the globulin plus fibrinogen were salted out with appropriate concentrations of sodium sulfate. The albumin remains in solution after the latter precipitation. The nitrogen of the fractions, the total nitrogen, and the non-protein nitrogen were determined by a microadaptation of the Kjeldahl method. Details of our system of analysis have been published elsewhere (12). Division of the plasma proteins into these fractions-albumin, globulin, and fibrinogen-has proved to be very useful in physiological studies and perhaps even more so in the practice of medicine, but the validity of this classification has been frequently questioned during the past twenty years ( 6 ) . The fractions which we call “albumins,” “globulins,” and “fibrinogen” are said to be artifacts, having no real existence in native plasma. We are asked, therefore, to abandon the old nomenclature and adopt a new one (6, 13). We believe that the proPresented a t the Nineteenth Colloid Symposium, which was held at the University of Colorado, Boulder, Colorado, June 18-20, 1942.
1118
ROBERT M. HILL AND VIRGINIA TREVORROW
priety of making this change in nomenclature depends upon the interpretation of available data, and, because of conflicting interpretations, it seems advisable to try to justify a point of view upon which we have expended a very considerable amount of time and work. In this attempt we shall use, as arguments from the literature, only a few of the most relevant experiments and some of the most cogent opinions based on experiment. Several degrees of opinion are expressed among those who favor abandoning the old classification. Some hold simply that the albumins and globulins are mixtures of proteins and that these names should be replaced by the names of the individual components of the mixtures (11, 13). Others hold that, although the albumins and globulins may be independent of each other, they form highly labile systems within themselves which undergo association and dissociation with such ease as to make characterization extremely difficult, if not impossible (22,23). The extreme opinion is that all of the protein of the plasma is present as one giant molecule, and that this giant molecule is held together by bonds so weak that they are disrupted by our methods of analysis. This is the point of view held by R. J . Block (3, 5 , 6). He has given the name “orosin” to this giant molecule, which he believes comprises all of the protein of the plasma, or other tissue. In 1938 Block wrote (6), “It seems that these proteins do not exist as a simple mixture, but as a system of reversible components so combined that the system behaves osmotically as a single substance;” and again, “experiments. . , throw strong doubt on the fundamental validity of attempting to classify the dissociable proteins as albumins, globulins, prolamines, etc. The experiments indicate that the soluble dissociable proteins do not exist in the tissues and organs as such, but are produced by the reagents employed in their preparation.” The first experimental work which supports the orosin theory was reported by Hardy (9) in 1903. He wrote, “The proteid of blood serum is electrically inactive-it will not move in a field. I t can however be made to move by appropriate treatment, and the character of the movement is such as could hardly be exhibited by a mixture of various colloids-in other words, the experiments suggest that only one proteid is present in serum and not several proteids.” More recently a series of papers from the laboratory of S. P. L. S0rensen in Copenhagen has given less direct support to this view. According to SOrensen, plasma protein is made up of a number of “coprecipitation systems” and each system consists of a number of polypeptides relatively weakly held together by secondary bonds. Under certain conditions there may be some exchange of polypeptides between systems. However, if we read SGrensen correctly, he considered that a t any moment of time the coprecipitation systems have separate exhence. In other words, the protein of the plasma would not behave “osmotically as a single substance.” The earlier work in Svedberg’s laboratory in Upsala added to the belief in the existence of an immense, vague, probably uncharacterizable protein molecule in the plasma. Ultracentrifugation has shown that by changing environmental conditions sumently, plasma proteins may be reversibly associated and dissociated.
1119
VARIATIOS I S PROTEIN CONCENTRATIOS I K PLASMA
In this country, R. J. Block has found a remarkable constancy in the basic amino acid content of whole plasma, and this, coupled with the evidence listed above, led him to propose the orosin theory. In Block’s words (4), ‘i. . . The term orosin has been introduced to designate the total coagulable protein of the serum.” This theory elicited the following protest from Madden and Whipple (19) in 1940: “Although recent evidence from Scandinavian, British and American laboratories renders it probable that these plasma protein fractions are all part of a single, variably bound protein system, we shall continue to speak of albumin, globulin and fibrinogen, for they do have a certain independent importance in biological reactions. Moreover, to be acceptable, new hypotheses of protein structure cannot be incompatible with this independence.” A very significant feature of this protest is the implied acceptance of the position that from the physicochemical point of view, a t least, the protein of the plasma may be looked upon as a “. . . single, variably bound protein system. . .” But do we need to accept this position? It seems to us that there is not only an abundance of physicochemical evidence, but also a considerable amount of physiological and pathological evidence for the existence of different independent protein units in native plasma. In 1930 Adair and Robinson (1) suggested that Hardy’s failure to observe any appreciable movement of the plasma proteins in an electric field may have been due to the low ratio of the charge to the mass of the molecules and to the ion atmosphere. In 1935 McFarlane (17), working in Svedberg’s laboratory, was able to separate the fractions of undiluted human plasma by means of a high potential applied through silver-silver chloride electrodes. He wrote, “We conclude from this experiment that the cataphoresis method is on the whole a satisfactory one for fractionating human sera.” This seems to dispose of the support given to the orosin theory by Hardy’s experiments. The analyst who determines the plasma protein fractions by the salting-out method is accused of dealing with artifacts. But the experiments of Sgjrensen upon which this criticism is, in part, based were carried out on dried serum, prepared by the method of Hardy and Gardiner (lo), a much more drastic treatment than salting out. In 1935, McFarlane (17), using the ultracentrifuge, found that . . drying cow serum by simple evaporation over PlOs,or drying and then ether extracting the powder or treating the serum with ammonium sulphate all give rise in varying degrees to this polydisperse protein.” This is very suggestive evidence that Sgjrensen’s work was carried out on artifacts, that part of his results were due to this fact, and that, therefore, his conclusions may not be applicable to native plasma. In a later publication of the same year, McFarlane (18) wrote, “The ultracentrifuge shows that in normal serum there are present three fractions each of which, if not completely homogeneous, can only be heterogeneous within very narrow limits.” And again (16), “It can be said from a consideration of the sedimentation constants that the average molecular weight of the lighter fraction corresponds in all mixtures approximately to that of albumin, and from considerations of the degree of boundary spreading that the fraction is probably homogeneous.” It seems that McFarlane’s experiments effectively nullify any arguments for
.
“.
1120
ROBERT M. HILL AND VIRGINIA TREVORROW
the orosin theory baaed on the work of Sorensen, and further show that the more recent experiments with ultracentrifugation are not out of harmony with the classification of the plasma proteins &s albumins, globulins, and fibrinogen. Further evidence is found in the ultrafiltration studies of Elford, Grabar, and Fischer (8). In 1936 they wrote, I ‘ . undoubtedly the albumin and globulin behave as distinct individuals in serum, the former existing mainly in the molecularly disperse condition whilst the globulin appears to be partly in an associated form and partly molecularly dispersed.” In 1937 Tiselius (25)wrote of studies with horse serum by the electr,ophoretic method: “With the aid of this method, serum as well as solution of serum globulin were found to contain several distinctly different components, which could be completely separated. Accordingly, serum is not a more or less continuous mixture, but contains &ell-defined protein fractions : albumin and three globulins a, p, and y.” In 1938 Stenhagen (24), using human serum, confirmed these findings of Tiselius. The protein of the tissue fluid and lymph is believed to have its origin in the plasma protein and reaches the lymph by filtration through the capillary wall (7). If this is true, the ratio of the albumin to globulin, the A/G ratio, &s determined by salting-out methods, should be the same in both plasma and lymph if the orosin theory correctly describes conditions existing in the plasma. If there is only one kind of protein molecule in the plasma, only one should filter through the capillary wall. If salting-out methods produce the albumin and globulin from orosin, they should produce them in the same ratio from the lymph as from the plasma. It would appear that the only escape from this conclusion lies in a possible specific association-dissociation activity of the capillary wall on the plasma protein, for which there seems to be no evidence. There are not many reports in the literature on the ratio of albumin to globulin in which determinations were made simultaneously on plasma and lymph. Those accumulated up to 1941 are discussed in the recent book by Drinker and Yoffey (7). In all cases the ratio of albumin to globulin is much higher in normal lymph than in the corresponding plasma, with the exception of liver lymph which is formed through capillary walls of exceptionally high permeability. This higher ratio in the lymph is interpreted by these authors to mean that the smaller albumin molecule filters through the capillary wall more readily than the larger globulin molecule. Very recently Leutscher (14),using the electrophoretic method, examined the plasma proteins and the proteins of other body fluids taken a t the same tiwe from patients with various diseases. He studied ascitic fluid in cases of cirrhosis, cardiac failure, tuberculosis, and carcinoma ; pleural fluid in cases of cardiac failure, glomerulonephritis, lobar pneumonia, tuberculosis, and Hogdkins disease; and pericardial fluid in one case of cardiac failure. In each case the proportion of albumin was higher in the pathological fluid. The amount of increase in the proportion of albumin ranged from 2 per cent of the total protein in a case of cirrhosis to 24 per cent of the total in a case of cardiac failure. Similar studies were made by Leutscher (15) on the proteins of plasma and urine
..
VARIATION IN P R ~ ~ E I CONCENTRATION N IN PLASMA
1121
in cases of nephrosis, nephritis, amyloid disease, and acute rheumatic fever. In these. studies the amount of increase in the proportion of albumin in the urine over that in the plasma, in terms of the total protein, ranged in three cases of nephrosis from 59 to 66 per cent, in two cases of terminal nephritis was 6.9 and 15.6 per cent, in one case of amyloid disease was 46.5 per cent, and in one case of rheumatic fever was 8.9 per cent. Thus, contrary to the requirements of the orosin theory, the proportion of albumin salted out of an ultrafiltrate of native plasma may be very much greater than the proportion of albumin salted out from the plasma itself. Fairly close agreement in the A/G ratio of the plasma was found between values obtained by the electrophoresis method and by the salting-out method of Howe, except in nephrosis, in which the A/G ratios were approximately twice as high by the salting-out method. The explanation for this seems to be that, in this disease, especially high values were found for globulin, and the globulin fraction, normally less than 10 per cent of the total, is found largely in the albumin fraction by salting-out methods (25). Melnick, Field, and Parnell (21) found that dilution of serum from healthy individuals with equal volumes of saline and then incubating a t 37°C. for 24 hr. did not change the analyses for albumin and globulin which were separated by a salting-out method. In a second series of experiments, these authors found that prolonged dialysis of normal sera against hypoproteinemic sera from patients with nephrosis did not change the A/G ratio of either. Again, contrary to the orosin theory, the low A/G ratios of the h l poproteinemic qera were not caused by changes of environment. In evaluating the in vitro experiments on plasma proteins in the light of their significance relative to the physiological activity of native plasma z n LWJO, special attention should be given to the extent of environmental change. -4change from pH 7.4 to pH 7.0 would be a very large in vivo change, but a small in vztro change. Similarly, a change of inorganic ions from 320 to 260 milliequivalents per liter in vivo would he a very large change, but of relatively slight importance in the studies of the effects of inorganic ion concentration on the proteins z n vitro. Most of the experimental association and dissociation of plasma proteins have been induced by changes in environmental conditions far outside of the possible physiological range. Thus, it appears that much of the argument for the association and dissociation of protein molecules and their interdependence loses its force when applied to native plasma in the living animal. When we consider the classification of the proteins from the point of view of the physiologist and the clinician, we must keep very clearly in mind that these men are thinking in terms of the protein present in the undisturbed animal and not of any extracted substance prepared by any artificial method VI hatsoever Consequently, if our methods of analysis measure some substance (or group of substances having closely related properties) which has independent existence in the plasma and corresponds in physiological function to what has been called “albumin,” then there is as yet insufficient argument for renaming this protein (or group of proteins). The same argument applies to the globulins and t o fibrinogen.
1122
ROBERT M. HILL AND VIRQINIA TREVORROW EXPERIMENTAL
The complete series of 566 determinations on 547 persons is made up of 303 determinations on males and 263 on females. These are distributed equally throughout the entire age sequence from birth to forty years, except in the range above twenty years, in which the females are predominant, and a t eleven years and below six months, where the number of males is in excess. No hospitalized subjects were used in this study. An attempt was made to rule out those who might present a clinical or subclinical variation in any of the protein fractions as a result of a recent immunization, malnutrition, dehydration, chronic or acute disease, or recent injury. All were examined by a physician either a t the time the blood was taken or shortly before and, to the best of our belief, were healthy persons. Most of the babies and younger children were from maternity hospitals or homes a t which they received regular medical care and in which the dietary was considered to be adequate. The adolescents were &dents a t West Denver High School. The adults were medical students and laboratory workers. Most of the blood samples were taken during the morning a t varying periods of time after meals. No consistent difference was found in any of the protein fractions between subjects who had fasted and other subjects of the same age. Similar findings have been reported by other workers. Blood samples were taken from the external jugular, the femoral, or the scalp veins in the small infants, and from the arm or hand veins in the larger children and adults. In taking the blood, a minimum amount of stasis was used. The blood sample was placed immediately in a tube containing a slight excess of dry heparin, mixed, and centrifuged in a stoppered tube as quickly as possible. Limitations of space prevent a detailed presentation of data; however, these have been analyzed by accepted statistical procedures (Mainland) (20) and the significant findings, arranged according to age groups, are shown in the figures.
Albumin The albumin fraction shows no significant variation between the sexes, or with the height, weight, or body surface of the subjects, but it does vary with the season of the year. During the first month of life there is no apparent change with age, but the variation between different babies of the same age is large. At about the end of the first month a rise in the albumin with increasing age becomes apparent. This continues until the “adult” level is reached between the ages of six months and one year, after which there is no further significant change with age. The mean albumin level for the first month of life is 3.79 g. per 100 cc. with a standard deviation of f 0.33 g. The mean “adult” level is 4.70 g. per 100 cc., with a standard deviation of + 0.32 g. The trend of values during the period of change is shown graphically in figure 1, together with the values for both younger and older persons. The spread covered by two standard deviations, including about 95 per cent of the cases, is very large for all ages. This spread is made up of two components, the true
VARIATION IN PROTEIN CONCENTRATION IN PLANA
1123
FIG.1. A scatter diagram of albumin, expressed as grama per 100 cc. of plasma, referred to age. The unbroken lines represent mean values; the area between the broken lines is that included between plus and minus two standard deviations from the mean.
RrCant
of Casr
"t
Albumin gn/roo c
FIG.2. Frequency polygons comparing winter and summer albumin values in 309 analyses on individuals over three years of age.
1124
ROBERT M. HILL AND VIRGIK1.4 TREVORROW
biologic variation and the variation caused by chance errors in the analytic procedure. The true biologic variation has been calculated as follows:
- (S.D.M)' = 40.32395' - 0.077052 Z/(S.D.Tjl
8.D.s
=7= f 0.314
Globulin Cm/toocc Plksma
x=MalQS.
.=Feinales.
ps-
1
I
I
I
I
I
I
I
I
-0 ~p*rsyr~z~&~wsm3h~lltlr. z4r 3qr:
qLp:
Vertical line rndicates a chanaoin age Scale. l
t q ~ yr:
l
~ q t :I Z ~ Z
I
MY 291:
I
40qr
A FIG.3. A scatter diagram of globulin, expressed as grams per 100 cc. of plasma, referred t o age (see legend of figure 1 ) .
05
-
a!-+,-.-
.. - - -, - _ '
*
8
VARIATION I N PROTEIN CONCENTRATION I N PLASMA
1125
publication (12). It is seen that the true biologic variation is much the larger component of the standard deviation. The albumin analyses have been studied with respect to seasonal variation. In the series of 127 babies up to one month of age, there is no significant difference in the plasma albumin level a t different seasons of the year. For persons over three years of age, 142 winter samples give a mean value of 4.83 ( S A T = f 0.30),and 167 summer samples show a mean of 4.59 (S.D.* = f 0.31). Al-
Fibrinogen gm./ioo cc.
as 0.4
-
0.30.2
-
0.1
-
I
,
.
I.
standaid deviation3 two standard deviations
1.5
2.0
2.5
3.0
Globulin
gm./ioo cc.
FIQ.5 . A scatter diagram of fibrinogen concentration referred to the corresponding globulin concentration in 259 samples of plasma from individuals over five years of age.
though this difference is not great, the probability that it would occur by chance alone is much less than 1 in 100. Figure 2 shows the frequency distribution of albumin values in the winter and summer groups. Bamtt, Sundermann, Doupe, and Scott (2) have shown that the total plasma protein concentration of human subjects drops during the transition from winter to summer. This is believed to be caused by a corresponding increase in plasma volume. As the warmer temperature is maintained, the protein concentration increases until the initial level is reached. During the transition from summer to
1126
ROBERT M. HILL AND VIRGINIA TREVORROW
winter, the reverse set of changes takes place. However, plasma volume changes are not the only factors determining the winter and summer concentrations of the plasma proteins, because globulin and fibrinogen do not show this seasonal variation. Globulin The plasma globulin concentration has been found to vary with age, aa shown in figure 3,but it does not vary with the sex or size of the person, or with the season of the year. During the first week of l i e the globulin shows a mean con-
Albumin
gm./ioo cc.
Sb s.0
4.S 4 .O
-Globulin, two standard deviations
- - Albumin, €wo standard deviations 1.s .
2.0 2.S Glob ul in
3.0
gm./iooec.
FIG.6. A scatter diagram of albumin concentration referred to the corresponding globulin concentration in 262 samples of plasma from individuals over five years of age.
centration of 1.66 g. per 100 cc. with a standard deviation of f 0.29. From the first to the fifth week, the globulin level falls to 1.31 g. per 100 cc. (S.D.= = f 0.25), where it is maintained until about the age of six months. At about six months, the globulin begins a gradual rise which continues until about the fourth year when the “adult” level of 2.03 g. per 100 cc. (S.D.= = f 0.34)is reached.
Fibrinogen The most striking feature of the findings with respect to fibrinogen is a wide scatter of the values in all groups. Figure 4 shows the distribution of the values
VARIATIOS I 5 PROTEIN COXCENTRATION I N PLASMA
1127
with respect to age. There is a slight tendency toward a higher mean value and greater variation a t birth than in the older age groups. After one month there is no significant change with age in the mean level of plasma fibrinogen. This value is 0.21 g. per 100 cc., with a standard deviation (total) of f 0.0590. If the orosin theory correctly describes the condition of protein in the plasma, then, in a large group of data such as we have collected, we should find a definite correlation between any two fractions. To test this we have prepared figures 5,6, and 7. Figure 5 is a scatter diagram of fibrinogen concentration referred to the corresponding globulin concentration in 259 samples of plasma from indi-
Fibrinooon
sn
I
b~
100
cc.
Q4
a3
I.
0.2
Q1
--Albumin, t w o standard dev ations - - FibrinoSan , two standard deviation,
4.0
4.5
5.0
5.5
Albumin
gm./ioo cc.
FIG. 7. A scatter diagram of fibrinogen concentration referred to the corresponding albumin concentration in 259 samples of plasma from individuals over five yeara of age.
viduals over five years of age. In the same way, figure 6 refers the albumin to the globulin concentration and figure 7 refers the fibrinogen to the albumin concentration. As can be seen from the figures, the correlation is slight. In only one case does a fibrinogen value above two standard deviations occur in a person having a globulin value above two standard deviations. Not a single corresponding high value for both fractions appears when albumin is referred to globulin, and when fibrinogen is referred to albumin. Corresponding low values (below two standard deviations) in both of any two fractions are entirely absent.
1128
ROBERT M. HILL AND VIRQINIA TREVORROW DISCUSSION
We have discussed our data in an earlier publication with reference to those presented by other authors, and such comparisons will be omitted here (26). In the introduction to this paper, we presented evidence from the literature for the independent existence in the plasma of the three major protein fractions, albumin, globulin, and fibrinogen. We have made 566 determinations of these fractions on 547 persons. If the orosin theory correctly describes the plasma proteins, correlations should be easily demonstrable between these fractions. We are unable to find such correlations. We believe, therefore, that, from the point of view of the clinician and the physiologist, the classification of the plasma proteins as albumins, globulins, and fibrinogen is still valid and we propose to use this classification until better reasons appear for discarding it. We realize that the fraction of the circulating plasma which we measure as albumin probably contains more than one component and that the fraction which we call globulin certainly contains several molecular species. These individual components may have specific functions, but they still have group importance. We also realize that our separations are not perfect. We hope that in the future better separations will be possible and that adequate analytical methods may be available for routine analysis of all of the protein components of plasma. But until that time, with Madden and Whipple, ‘ I . . . we shall continue to speak of albumin, globulin and fibrinogen, for they do have a certain independent importance in biological reactions.” And we also believe with them that, ‘ I . to be acceptable, new hypotheses of protein structure cannot be incompatible with this independence.”
. .
SUMMARY
1
1. A total of 566 analyses of plasma albumin, globulin, and fibrinogen have been made on 547 persons from birth to thirty-nine years of age. The changes with age are defined. 2. The plasma albumin concentration in persons over three yean of age is higher in the winter than in the summer months. No seasonal variations appear in the other fractions. 3. The albumin, globulin, and fibrinogen concentrations vary independently. This independent variation is not in harmony with the orosin theory of the structure of plasma proteins. REFERENCES (1) ADAIR,G . S.,AND ROBINSON, M. E . : Biochem. J. 24, 1864 (1930). (2) BAZETT,H.C., SUNDERLAND, F . W., DOUPE,J., AND SCOTT,J. C.: Am. J. P b s i o l . lW,69 (1940). (3) BLOCK,R. J.: J. Biol. Chem. 106, 455 (1934). (4) BLOCK, R. J.: Yale J. Biol. Med. 7,235 (1934-35). (5) BLOCK, R . J.: Yale J. Biol. Med. 9, 445 (1936-37). (6) BLOCK,R. J.: In Chemistry of the Amino Acids and Proteins, edited by C. L. A. Schmidt. Charles C. Thomas, Springfield, Illinois (1938). (7) DRINKER,C. K.,AND YOFFEY,J. M.: Lymphatics, Lymph and Lymphoid Tissues. Harvard University Press,Cambridge (1941).
ELECTROPHORETIC PATTERN IN EXTRACTS OF POLLENS
1129
(8) ELFORD, W. J., GRABAR, P., AND FISCHER, W.: Biochem. J. SO, 92 (1936). (9) HABDY,W. B.: J. Physiol. 18, XXVI P (1903). (10) HARDY,W.B., AND GARDINER, S.: J. Physiol. 40, LXVIII P (1910). (11) HEWITT, L. F.: Biochem. J. 91,360 (1937). AND TREVORROW, V.: J. Lab. Clin. Med. 1 , 1838 (1941). (12) HILL,R . M., (13) K'YLIN,E . : I n Die Eiweisskdrper des Bhtplasmas, by H. Bennhold, E. Kylin, and S. RusenyBk. T. Steinkopff, Dresden and Leipzig (1938). (14) LEUTBCRER, L. A,: J. Clin. Investigation 10, 99 (1941). L. A.: J. Clin. Investigation 19, 313 (1940). (15) LEUTBCRER, A. S.:Biochem. J. 18,407 (1935). (16) MCFARLANE, (17) MCFARLANE, A. S.: Bioohem. J. 29,660 (1935). A. S.: Biochem. J. 18. 1209 (1935). (18) MCFARLANE, S. C., AND WHIPPLE,G. H.: Physiol. Rev. 20,194 (1910). (19) MADDEN, (20) MAINLAND, D.: The Treatment of Clinical and Laboratory Data. Oliver and Boyd, London (1938). (21) MELNICK, D., FIELD, H., A m PARNELL, C. G., J R . : Arch. Internal Med. 66,295 (1910). N , P. L.: J. Am. Chem. SOC. 47, 457 (1925). (22) S ~ R E N S E S. (23) S ~ R E N S E S. N ,P. L.: Kolloid-2. 69, 102 (1930). E . : Biochem. J. SZ, 714 (1938). (24) STENHAGEN, (2.5) TISELIUS,A,: Biochem. J. 31, 1464 (1937). (26) TREVORROW, V.,KASER,M., PATTERSON, J. P., AND HILL, R. hf,: J. Lab. Clin. hled. a7, 471 (1942).
A GEXERAL ELECTROPHORETIC PATTERN IN EXTRACTS OF POLLEKS CAUSING HAY FEVER',' HAROLD A. ABR.4MSON, DAK H. MOORE,
AND
HENRIETTE H . GETTNER
The Electrophoresis Laboratory and the Department of Physiology, Columbia University, and the Medical Service of D r . George Baehr and the Laboratories of the Mount Sinai Hospital, New York City, New York Received August 4 , 19.42
In aqueous extracts of the pollen of both giant and dwarf ragweed a t about pH 7.0 there is a slow-moving major colorless component (US) which is highly skin reactive and which produces hay fever and asthma (1). The component in giant ragweed, Trifidin (USG), and the component in dwarf ragweed, Artefolin (USD), were electrophoretically isolated and ultracentrifuged. From the diffusion and sedimentation constants, the molecular weight of each component was calculated to be about 5OOO. There were also usually present in curves for the whole pollen extracts from four to six minor pigmented boundaries as well as the US component. The fastest moving pigments were biologically active and showed skin reactions both by direct tests and by indirect tests (passive transfer). Because of the small molecular size of the components isolated, the 1 Presented a t the Nineteenth Colloid Symposium, which was held a t the University of Colorado, Boulder, Colorado, June 18-23, 1942. This investigation has been aided by a grant from the Josiah Macy, Jr., Foundation. f
