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The major electrophoretic component of giant ragweed extracts: Molecular weight, chemical and biologic characteristics
THE MAJOR ELECTROPHORETIC COMPONENT OF GIANT .RA(:WEE L, EXTRACTS : MOLECULAR WEIGHT, CHEMICAL AND BTOLOGIC CHARACTERISTICS* FRANKLIN
A.
STEVENS,M.D., DAN MOORE,PH.D., NEW YORK, N. -I’-.
SNI) TOSCA GEI,STON,
BRAMSON and his a.ssociateslhave described t.he electrophoretic patterns of whole extracts of ragweed, certain grasses,and tree pollens in a series of A publications. In each of these extracts they found a slow-moving, unpigmented fraction, which after separation in the cell was found extremely active when tested in the skin of sensitive patients, and also a rapidly moving pigment of minor specific biologic importance. Between these two, the slowest,and the most motile fract,ions, were boundaries of other pigmented substances, all moving toward the negative portion of the cell. By ultracent,rifugation they calculated that the slow or major component separated electrophoretically from ragweed soIutions had an approximate molecular weight of 5,000.2 This fraction gave most of the tests considered chaiacteristics of protein. Saniga? had concluded previously from ultracentrifugal studies of ragweed ext,racts that they were molecularly heterogeneousand comprised mostly7 of molecules of weights less than 17,000. Data indicating that the proteins in ragweed extracts gradually break down into fragments of lesser magnitude have been published previously”; a.t 87” (1. the nitrogen in ragweed extracts precipit,ated by full and half saturation with Na, SO, decreased decidedly within a period of a few days. Stull, Sherman, and Wing4 have also found that active “protein” fractions precipitated by (NH,),SO, were retained within a dialyzing membrane. These experiment,s suggest that molecules larger than 5,000 molecular weight probably exist in these extracts and possibly could be separated from sma,ller fragments by prolonged dialysis, The rapidly moving pigment in ragweed extracts described by Abramson has been identified as isoquercitrin5 Without drastic methods isolated isoquercitrin could not be freed from biologically active nitrogen, but the nitrogen could be reduced by gentle treatment to a point where reactions in sensitive patients were no longer obtained. The following data are concerned with the material retained within a Visking membrane with a calculated average pore size of 2.5 Angstrom units. EXPERIMENTAL
Giant ragweed pollen previously washed in ether was extracted in distilled water. The extract was washed, dialyzed, and concentrated in a Visking membrane with buffer solution at pH 8.4 under a pressure of 50 mm. of mercury. Portions of the concentrate within the sac were removed after 10 days of dialysis and dilutions made with buffer mixture were compared as to electrophoretic pattern with the original extract and the dialysate containing 50 mg. N per cent (Fig. 1). The pollen used in these experiments was supplied by Wyeth, Incorporated. *From the Department of Medicine and the Electrophoretlc Laboratory, Columbia University College of Physicians and Surgeons, and the Presbyterian Hospital, New York, N. Y. 356
STEVENS ET AL. : ELRCTROPHORETIC
COMPONENT
OF RAGWEED EXTRACT
357
A second lot of pollen was treated in the same manner except that M/15 phosphate mixture (pH 7.4) was used throughout. Portions of the concentrate and dialysate were precipitated by full and half saturation with Na, SO, and with 5 per cent trichloracetic acid, the nitrogen was determined in each precipitate by the micro-Kjeldahl method, and the percentages of precipitable nitrogen in these catagories were calculated for the total material within and for that without the membrane. Sterile dilutions of the original extract, the concentrate, and the dialysate were also prepared for skin testing, and the threshold of the activity of each preparation, based on nitrogen content, was determined.
Fig. l.-Electrophoretic patterns of ra eed extracts: a, concentrated dialysate, 60 mg. N/100 c.c., 61 minutes, pH 8.4., mobility of 8gw ow component 0.8 cm. X 10-E volt second; b, concentrate within the dialyzing sac, 86.6 mg. N/100 c.c., 180 minutes, pH 8.4. slow component 6.6 cm. x 10-S volt second: I?, untreated extract, 50 mg. N/100 c.c., 60 minutes, pH 8.4, slow component 0.6 cm. X 10-o volt second.
The remainder of the concentrate dialyzed overnight against M/15 phosphate buffer mixture (pH 7.4) was subjected to ultracentrifugal analysis. Sedimentation constants were determined in an air-driven vacuum centrifuge,6 the sedimentation boundary being recorded by the scanning method of Longsworth.’ Two analyses were made at room temperature. Since the temperature of the rotor spinning at 48,000r.p.m. rose about 1.2Oeach hour, it was possible
358
THE
JOURNAL
OF ALLERGY
to estimate the average temperature over any desired time interval in the run. All values of sedimentation constants were reduced to conditions of pure water at ZOO. Diffusion experiments were carried out in a Tiselius cell in M/15 phosphate mixture at lo. The following data were obtained : D,,, 7.1 x lo-? cm.2 per second; &,, 1.3 x lo-l3 cm. per second per unit field; assumedpartial specific volume, 0.75; frictional ratio, f/f,,,, 1.72; molecular weight, M, 17,800, and eccentricity, l/e, 14. The material appeared to be homogeneous. DISCUSSION
When the extract of giant ragweed was dialyzed, approximately 85 per cent of the nitrogen passed through the membrane (Table I). The concentrated material within the sausage,light brown in color, was almost one-half precipitated by saturated Na, SO, (Table II), while only 3 per cent of the nitrogen in the dialysate was precipitated by this reagent. It was anticipated that large molecules, if present, would be retained and precipitated in t,his manner, while those which might be smaller and not precipitable would escape. On the other hand, although very much diluted, slightly more nitrogen in precipitable form was found in the dialyzate than in the concentrate (Table II). Several possible explanations may be given for the escape of this material. The molecular size of the “protein” precipitated by saturation with Na, SO, might be just on the border line of the permeability of the membrane, the pores may have been enlarged by the pressure applied over a period of several days, or as subsequent data suggest, the molecule may be elongated. In some experiments, when extracts were dialyzed at 4” for a period of 10 to 15 days, isoquercitrin crystals actually formed within the substance of the membrane, causing slow leaks. By correlating the ultracentrifugal and diffusion data it was found that the molecules composing the material within the membrane had molecular weights of TABLE
DAYS 3 3 7 IO
10
I
INITIAL (MC.) 25.4 65.2 80.6 99.4
99.4
I. N
PARTITION
OF N
CONCENTRATED (Ma. %) 3.6 14 11.5 19 7.1 8 5.9 6 6.0 6
IN EXTRACTS N
BY DIALYSIS*
DIALYZED N (MO. 96) 20.5 82 50.0 76 71.2 88 86.0 86 87.0 X7
RECOVERED N (MG.) 24.1 61.2 78.3
93.5t 94.5t
*In this table, as well as subsequent tables and data, the material within the Visking sausage may be designated as “concentrate” and that without as “dialysate.” tThese totals include N in an insoluble precipitate in the concentrated extract, 1.5 and 1.6 mg. respectively, in the last 2 experiments.
about 18,000 and appeared to be elongated with a ratio of length to breadth of 14 to 1. In a previous publication the rapidly moving pigment described by Abramson was identified as isoquercitrin. This study is concerned only with the slowly moving, unpigmented fraction. A comparison of the patterns of whole extract (Fig. 1)) the dialysate, and the concentrated material resulting from long-continued dialysis of the extract shows that a component with the same mobility as that described by Abramson occurs in all 3 and is the only component occurring
STEVENS
ET
ELECTROPHORETIC
AL.:
COMPONENT
OF RAGWERD
359
EXTRACT
in the pattern of the material within the bag. From our observations and those of Abramson it would appear that this component in the pattern of the whole extract is largely a mixture of nitrogen containing molecules of various weights, In a mixture of molecules comprised principally of all with the same mobility. those of low weight not precipitable by saturating with Na, SO, (Table II), the sedimentation and diffusion boundaries of the sparse molecules of greater weight, such as 18,000, might be obscured and readily overlooked in analyses of whole extracts such as those used by Abramson. From data in a previous communication it was believed that the proteins in the extracts were constantly breaking down into fragments of lesser size, a conclusion that can readily be harmonized with the observations presented here. TABLE
II.
N
PARTITION
OF CONCENTRATE
AND DIALYSATE
BY PROTEIN
DIALYSATE
CONCENTRATE PPT. N (MG. %) 0.3 0.08 25
TOTAL N (MG.1
TOTAL N (MG-)
M Saturated Na, SO, (37") 5% Trichloracetic acid lie Saturated Na, SO, l/l Saturated Na, SO,
0.3 0.6 0.6
0.08 0.167 0.27
PRECIPITANTS
4.85 4.85 9.70 9.70
25 28 45
PPT. N (MG. %) 0.08 1.6
0.08 0.2 0.3
1.6 2.0 3.0
No study of fractions of pollen extracts is complete without correlative Dilutions of the whole observations on the biologic activity of the fractions. extracts, of the material within the dialyzing bags, and of the dialysates of extracts were prepared, using total nitrogen as a standard, so that the thresholds of the activity of the 3 preparations could be compared by testing sensitive patients. Since a tenfold difference between dilutions is usually necessary in cutaneous testing to provoke responses with solutions of different strengths by which the dilutions can be clearly distinguished from each other, the dilutions of these preparations were made in series with each successive dilution 10 times weaker than that preceding. The strongest in each series contained 10 mg. of N in 100 CC. The material contained within the membrane gave more nearly TABLE
2
PATIENT E-
R: A: T. C.
p”: %. c.
III.
CUTANEOUS
ACTIVITY
THRESHOLD REACTION OF EXTRACT (MG. N/loo C.C.) 10.0 1 1.0 ( 0.1 ( 0.01 2t -2+ --
2t
f
lt 3;
2t
equal reactions in all patients patients reacted more strongly the other two. The dialysate stronger dilution to obtain a centrate or the whole extract.
i-
--
OF EXTRACT,
CONCENTRATE,
THRESHOLD REACTION OF CONCENTRATE (MG. ~/lo0 c.c.) 10.0 1 3.0 1 0.1 1 0.01 3t -2t --
3t 2t
AND DIALYSATE
THRESHOLD REACTION OF DIALYSATE (MG. N/loo C.C.) 10.0 1 1.0 1 0.1 1 0.01 3;
it
4
4t
i;
3+
2-t
2+
lt
__
ii --
--
it.
-_
than did the whole extracts or the dialysates, and to a weaker dilution of this preparation than of was the poorest cutaneous antigen, requiring a minimal reaction than was needed with the conSome patients who reacted to the concentrate and
360
THE
JOURNAL
OF
ALLERGY
the whole extract showed no reaction to the strongest solution of dialysat,e. Although some of the higher molecular material found predominately concentrated in the membrane appeared also in the dialysate, apparently this more active material was diluted with less a,ntigenic nitrogenous fractions until it was no longer potent. The reactions of several of the patients tested have been charted in Table III. Patients with hay fever have been successfully immunized with the concentrates From preliminary observations, per unit of total nitrogen, injections of the concentrates of ragweed pollen extracts appear superior to injections of the whole extracts for t,he prevention of hay fever. The dialysates may give reasonably satisfactory skin tests but still be inferior immunizing agents. Methods useful in obtaining quantities of stable material similar to the concentrate sufficient to immunize a large number of patients have only recently bean devised. REFERENCES
II 1. Abramson, H. A.: Chemical, Physical and Immunologic Properties of Electrophoretically Purified Pollen Extracts, Ann. Allergy 5: 19, 1947. l%47. Electrophoretic and Ultracentrifugal 2. Abramson, H. A., Moore, D. H., and Gettner, H. H.: Analysis of Hay-Fever-Producing Component Ragweed Pollen Extract, J. Phys. Chem. 46: lQ2. 192, 1942. 1942. The Quantitative Changes in Various Fractions of the Precipitable Nitrogen 3. Stevens, F. A.: in Ragweed Extracts at 37” c'., 90, lY47. 1947. C., J. ALLERGY 18: YU, Antigenic Fractions in Ragweed Pollen, J. 4. Stull, A., Sherman, W. B., and Wing, W. M.:
ALLERGY 13: 537, 1942.
The Isolation of Isoquercetrin From Giant 5. Stevens, F. A., Moore, D., and Baer, H.: Ragweed Pollen; the Electrophoretic Pattern and Biologic Activity of the Pigment. (To (To be be pl,hli.hd published.) \ Hormone Studies With Ultracentrifuge : III, 6. Chiles, J. A., JI Jr., , and Severinghaus, A. E.: Application of the Toepler Schlieren Method to the Analytic Ult,racentrifuge, Rev. T Scient. Instruments 11: 1 71, 1940. Modification of the Schlieren Method for Use in Electrophoretic 7. Longsworth, L. G.: Modi Some Improvements for Analysis, J. Am. Chem. Soe. 1939; Moore, D. H.: SOC. 61: 529, ! Rev. 14: 295, 1943. the Analytic Ultracentrifuge, Rev Scient. Instruments Ultracentrifugal Studies of Ragweed Extracts, J. Franklin Inst. 230: Stuc 8. Sanigar, E. B.: 781, 1940.
A.
STEVENS,M.D., DAN MOORE,PH.D., NEW YORK, N. -I’-.
SNI) TOSCA GEI,STON,
BRAMSON and his a.ssociateslhave described t.he electrophoretic patterns of whole extracts of ragweed, certain grasses,and tree pollens in a series of A publications. In each of these extracts they found a slow-moving, unpigmented fraction, which after separation in the cell was found extremely active when tested in the skin of sensitive patients, and also a rapidly moving pigment of minor specific biologic importance. Between these two, the slowest,and the most motile fract,ions, were boundaries of other pigmented substances, all moving toward the negative portion of the cell. By ultracent,rifugation they calculated that the slow or major component separated electrophoretically from ragweed soIutions had an approximate molecular weight of 5,000.2 This fraction gave most of the tests considered chaiacteristics of protein. Saniga? had concluded previously from ultracentrifugal studies of ragweed ext,racts that they were molecularly heterogeneousand comprised mostly7 of molecules of weights less than 17,000. Data indicating that the proteins in ragweed extracts gradually break down into fragments of lesser magnitude have been published previously”; a.t 87” (1. the nitrogen in ragweed extracts precipit,ated by full and half saturation with Na, SO, decreased decidedly within a period of a few days. Stull, Sherman, and Wing4 have also found that active “protein” fractions precipitated by (NH,),SO, were retained within a dialyzing membrane. These experiment,s suggest that molecules larger than 5,000 molecular weight probably exist in these extracts and possibly could be separated from sma,ller fragments by prolonged dialysis, The rapidly moving pigment in ragweed extracts described by Abramson has been identified as isoquercitrin5 Without drastic methods isolated isoquercitrin could not be freed from biologically active nitrogen, but the nitrogen could be reduced by gentle treatment to a point where reactions in sensitive patients were no longer obtained. The following data are concerned with the material retained within a Visking membrane with a calculated average pore size of 2.5 Angstrom units. EXPERIMENTAL
Giant ragweed pollen previously washed in ether was extracted in distilled water. The extract was washed, dialyzed, and concentrated in a Visking membrane with buffer solution at pH 8.4 under a pressure of 50 mm. of mercury. Portions of the concentrate within the sac were removed after 10 days of dialysis and dilutions made with buffer mixture were compared as to electrophoretic pattern with the original extract and the dialysate containing 50 mg. N per cent (Fig. 1). The pollen used in these experiments was supplied by Wyeth, Incorporated. *From the Department of Medicine and the Electrophoretlc Laboratory, Columbia University College of Physicians and Surgeons, and the Presbyterian Hospital, New York, N. Y. 356
STEVENS ET AL. : ELRCTROPHORETIC
COMPONENT
OF RAGWEED EXTRACT
357
A second lot of pollen was treated in the same manner except that M/15 phosphate mixture (pH 7.4) was used throughout. Portions of the concentrate and dialysate were precipitated by full and half saturation with Na, SO, and with 5 per cent trichloracetic acid, the nitrogen was determined in each precipitate by the micro-Kjeldahl method, and the percentages of precipitable nitrogen in these catagories were calculated for the total material within and for that without the membrane. Sterile dilutions of the original extract, the concentrate, and the dialysate were also prepared for skin testing, and the threshold of the activity of each preparation, based on nitrogen content, was determined.
Fig. l.-Electrophoretic patterns of ra eed extracts: a, concentrated dialysate, 60 mg. N/100 c.c., 61 minutes, pH 8.4., mobility of 8gw ow component 0.8 cm. X 10-E volt second; b, concentrate within the dialyzing sac, 86.6 mg. N/100 c.c., 180 minutes, pH 8.4. slow component 6.6 cm. x 10-S volt second: I?, untreated extract, 50 mg. N/100 c.c., 60 minutes, pH 8.4, slow component 0.6 cm. X 10-o volt second.
The remainder of the concentrate dialyzed overnight against M/15 phosphate buffer mixture (pH 7.4) was subjected to ultracentrifugal analysis. Sedimentation constants were determined in an air-driven vacuum centrifuge,6 the sedimentation boundary being recorded by the scanning method of Longsworth.’ Two analyses were made at room temperature. Since the temperature of the rotor spinning at 48,000r.p.m. rose about 1.2Oeach hour, it was possible
358
THE
JOURNAL
OF ALLERGY
to estimate the average temperature over any desired time interval in the run. All values of sedimentation constants were reduced to conditions of pure water at ZOO. Diffusion experiments were carried out in a Tiselius cell in M/15 phosphate mixture at lo. The following data were obtained : D,,, 7.1 x lo-? cm.2 per second; &,, 1.3 x lo-l3 cm. per second per unit field; assumedpartial specific volume, 0.75; frictional ratio, f/f,,,, 1.72; molecular weight, M, 17,800, and eccentricity, l/e, 14. The material appeared to be homogeneous. DISCUSSION
When the extract of giant ragweed was dialyzed, approximately 85 per cent of the nitrogen passed through the membrane (Table I). The concentrated material within the sausage,light brown in color, was almost one-half precipitated by saturated Na, SO, (Table II), while only 3 per cent of the nitrogen in the dialysate was precipitated by this reagent. It was anticipated that large molecules, if present, would be retained and precipitated in t,his manner, while those which might be smaller and not precipitable would escape. On the other hand, although very much diluted, slightly more nitrogen in precipitable form was found in the dialyzate than in the concentrate (Table II). Several possible explanations may be given for the escape of this material. The molecular size of the “protein” precipitated by saturation with Na, SO, might be just on the border line of the permeability of the membrane, the pores may have been enlarged by the pressure applied over a period of several days, or as subsequent data suggest, the molecule may be elongated. In some experiments, when extracts were dialyzed at 4” for a period of 10 to 15 days, isoquercitrin crystals actually formed within the substance of the membrane, causing slow leaks. By correlating the ultracentrifugal and diffusion data it was found that the molecules composing the material within the membrane had molecular weights of TABLE
DAYS 3 3 7 IO
10
I
INITIAL (MC.) 25.4 65.2 80.6 99.4
99.4
I. N
PARTITION
OF N
CONCENTRATED (Ma. %) 3.6 14 11.5 19 7.1 8 5.9 6 6.0 6
IN EXTRACTS N
BY DIALYSIS*
DIALYZED N (MO. 96) 20.5 82 50.0 76 71.2 88 86.0 86 87.0 X7
RECOVERED N (MG.) 24.1 61.2 78.3
93.5t 94.5t
*In this table, as well as subsequent tables and data, the material within the Visking sausage may be designated as “concentrate” and that without as “dialysate.” tThese totals include N in an insoluble precipitate in the concentrated extract, 1.5 and 1.6 mg. respectively, in the last 2 experiments.
about 18,000 and appeared to be elongated with a ratio of length to breadth of 14 to 1. In a previous publication the rapidly moving pigment described by Abramson was identified as isoquercitrin. This study is concerned only with the slowly moving, unpigmented fraction. A comparison of the patterns of whole extract (Fig. 1)) the dialysate, and the concentrated material resulting from long-continued dialysis of the extract shows that a component with the same mobility as that described by Abramson occurs in all 3 and is the only component occurring
STEVENS
ET
ELECTROPHORETIC
AL.:
COMPONENT
OF RAGWERD
359
EXTRACT
in the pattern of the material within the bag. From our observations and those of Abramson it would appear that this component in the pattern of the whole extract is largely a mixture of nitrogen containing molecules of various weights, In a mixture of molecules comprised principally of all with the same mobility. those of low weight not precipitable by saturating with Na, SO, (Table II), the sedimentation and diffusion boundaries of the sparse molecules of greater weight, such as 18,000, might be obscured and readily overlooked in analyses of whole extracts such as those used by Abramson. From data in a previous communication it was believed that the proteins in the extracts were constantly breaking down into fragments of lesser size, a conclusion that can readily be harmonized with the observations presented here. TABLE
II.
N
PARTITION
OF CONCENTRATE
AND DIALYSATE
BY PROTEIN
DIALYSATE
CONCENTRATE PPT. N (MG. %) 0.3 0.08 25
TOTAL N (MG.1
TOTAL N (MG-)
M Saturated Na, SO, (37") 5% Trichloracetic acid lie Saturated Na, SO, l/l Saturated Na, SO,
0.3 0.6 0.6
0.08 0.167 0.27
PRECIPITANTS
4.85 4.85 9.70 9.70
25 28 45
PPT. N (MG. %) 0.08 1.6
0.08 0.2 0.3
1.6 2.0 3.0
No study of fractions of pollen extracts is complete without correlative Dilutions of the whole observations on the biologic activity of the fractions. extracts, of the material within the dialyzing bags, and of the dialysates of extracts were prepared, using total nitrogen as a standard, so that the thresholds of the activity of the 3 preparations could be compared by testing sensitive patients. Since a tenfold difference between dilutions is usually necessary in cutaneous testing to provoke responses with solutions of different strengths by which the dilutions can be clearly distinguished from each other, the dilutions of these preparations were made in series with each successive dilution 10 times weaker than that preceding. The strongest in each series contained 10 mg. of N in 100 CC. The material contained within the membrane gave more nearly TABLE
2
PATIENT E-
R: A: T. C.
p”: %. c.
III.
CUTANEOUS
ACTIVITY
THRESHOLD REACTION OF EXTRACT (MG. N/loo C.C.) 10.0 1 1.0 ( 0.1 ( 0.01 2t -2+ --
2t
f
lt 3;
2t
equal reactions in all patients patients reacted more strongly the other two. The dialysate stronger dilution to obtain a centrate or the whole extract.
i-
--
OF EXTRACT,
CONCENTRATE,
THRESHOLD REACTION OF CONCENTRATE (MG. ~/lo0 c.c.) 10.0 1 3.0 1 0.1 1 0.01 3t -2t --
3t 2t
AND DIALYSATE
THRESHOLD REACTION OF DIALYSATE (MG. N/loo C.C.) 10.0 1 1.0 1 0.1 1 0.01 3;
it
4
4t
i;
3+
2-t
2+
lt
__
ii --
--
it.
-_
than did the whole extracts or the dialysates, and to a weaker dilution of this preparation than of was the poorest cutaneous antigen, requiring a minimal reaction than was needed with the conSome patients who reacted to the concentrate and
360
THE
JOURNAL
OF
ALLERGY
the whole extract showed no reaction to the strongest solution of dialysat,e. Although some of the higher molecular material found predominately concentrated in the membrane appeared also in the dialysate, apparently this more active material was diluted with less a,ntigenic nitrogenous fractions until it was no longer potent. The reactions of several of the patients tested have been charted in Table III. Patients with hay fever have been successfully immunized with the concentrates From preliminary observations, per unit of total nitrogen, injections of the concentrates of ragweed pollen extracts appear superior to injections of the whole extracts for t,he prevention of hay fever. The dialysates may give reasonably satisfactory skin tests but still be inferior immunizing agents. Methods useful in obtaining quantities of stable material similar to the concentrate sufficient to immunize a large number of patients have only recently bean devised. REFERENCES
II 1. Abramson, H. A.: Chemical, Physical and Immunologic Properties of Electrophoretically Purified Pollen Extracts, Ann. Allergy 5: 19, 1947. l%47. Electrophoretic and Ultracentrifugal 2. Abramson, H. A., Moore, D. H., and Gettner, H. H.: Analysis of Hay-Fever-Producing Component Ragweed Pollen Extract, J. Phys. Chem. 46: lQ2. 192, 1942. 1942. The Quantitative Changes in Various Fractions of the Precipitable Nitrogen 3. Stevens, F. A.: in Ragweed Extracts at 37” c'., 90, lY47. 1947. C., J. ALLERGY 18: YU, Antigenic Fractions in Ragweed Pollen, J. 4. Stull, A., Sherman, W. B., and Wing, W. M.:
ALLERGY 13: 537, 1942.
The Isolation of Isoquercetrin From Giant 5. Stevens, F. A., Moore, D., and Baer, H.: Ragweed Pollen; the Electrophoretic Pattern and Biologic Activity of the Pigment. (To (To be be pl,hli.hd published.) \ Hormone Studies With Ultracentrifuge : III, 6. Chiles, J. A., JI Jr., , and Severinghaus, A. E.: Application of the Toepler Schlieren Method to the Analytic Ult,racentrifuge, Rev. T Scient. Instruments 11: 1 71, 1940. Modification of the Schlieren Method for Use in Electrophoretic 7. Longsworth, L. G.: Modi Some Improvements for Analysis, J. Am. Chem. Soe. 1939; Moore, D. H.: SOC. 61: 529, ! Rev. 14: 295, 1943. the Analytic Ultracentrifuge, Rev Scient. Instruments Ultracentrifugal Studies of Ragweed Extracts, J. Franklin Inst. 230: Stuc 8. Sanigar, E. B.: 781, 1940.