- Email: [email protected]
Immunologic studies of Caddis fly
Her ORIGINAL
Physical
Sidney
NO.
1
JANUARY-FEBRUARY,
1963
ARTICLES
IMMUNOLOGIC III.
v 0 I‘ . 34,
STUDIES
and Chemical
Shulman,
Ph.D.,*
Paul
OF CADDIS
Characterization Bronson,
and
FLY
of the Major Carl
E.
Arbesman,
Antigen &l.D.,
Buffalo,
N.
Y
of Caddis fly bodies, having potent allergenic activity, have been prepared. These have been fractionated by zone electrophoresis to yield >I selected product, termed Pool 2, containing a considerably higher specific allcrgenicit,y than any of the other fractions. This material has now been collected from repeated electrophoretic separations, and these have been combined in order to give enough material for chemical and physical characterization. These properties are reported here.
E
XTRACTS
MATERIALS
AND
METHODS
Caddis fly extract was prepared by the direct method, as described in the first paper of this series.l Electrophoretic separation was then carried out on these extracts by means of curtain electrophoresis, using the procedures previously described.“, 3 A volume of 10 ml. was applied for each run. The resulting sets of 32 tubes were examined for protein concentration, and the tubes within the group corresponding to Pool 2 were pooled and saved. All other fractions were discarded. For the studies reported here, it was essential to have the material considerably more concentrated than had been necessary for evaluation of the allergenicity. It was found very useful to use the method of dry sucroseosmosis for this purpose. The sample was placed in a tied dialysis bag (Visking From the Departments of Bacteriology and Immunology and of Medicine, University of Buffalo School of Medicine, and the Allergy Research Laboratory of the BufPalo General Hospital, Buffalo. N. Y. This investigation was supported in part by Research Grants, E-1303 from the National Institute of Allergy and Infectious Diseases, and A-4195 from the National Institute of Arthritis and Metabolic Diseases, U. S. Public Health Service, Bethesda, Md. Received for publication July 30, 1962. *Recipient of a Research Career Development Awar (GM-K3-1377) from the U. S. Public Health Service.
2
SHULMAN,
BBONSON,
ARBESMAN
January-February,
J. Allergy 1963
cellophane) and put in a glass dish. Dry sucrose was poured on and around it. After several hours, the volume of solution in the bag had decreased to about one half, or less. The wet sucrose was washed away and the bag was tied again at the smaller volume. It could then be dialyzed against a saline or buffer solution, as desired. Analyses by ultracentrifuge and by optical electrophoresis were done and computed by standard methods, using the Spinco Models E and H, respectively. Nitrogen analysis was conducted by a semi-micro Kjeldahl method. Titration was performed with 0.01 N HCl, using methyl purple as an indicator. Light absorption studies were made with a Beckman DU spectrophotometer. Hexose was determined by the orcinol method, using a galactose-mannose standard, as described by Winzler.4 1.200
1.000
-
a c z 0
0.000
-
;
0.600
-
0.400
-
0.200
-
u Ti 0
0.000
Fig.
1.4ptical
density
plotted
’ 240
against
’
’ 260 Wavelength wave
’
’ 280 (mp)
length
’
for
: 10
a direct
Caddis
fly
extract.
RESULTS
Ultraviolet Spectrum.-A sample of the original Caddis fly extract was examined for its light absorption over the range of wave lengths from 220 to 290 rnp. The spectrum is shown in Fig. 1. The anticipated maximum for protein (at about 280 mp) is poorly resolved on the low wave length side, presumably because of the presence of an appreciable amount of nucleic acids and polynucleotides, absorbing maximally at 260 mp. The protein absorption seems to be maximal at about 275 mp. A spectrum of t.his type for the Pool 2 preparation is shown in Fig. 2. It is somewhat similar to that for the whole extract, although a better degree of resolution at 280 rnp is now evident; the polynucleotide absorption is proportionately decreased. Extinction Coefficients and Nitrogen Content,.-To explore the question of whether or not this purified substance is a protein, or whether perhaps partly
Volume 34 Number I
IMMUNOLOGIC
STUDIES
OF
(IADDIS
FL‘T-.
III
3
protein, a nitrogen analysis was carried out. To make this information meaningful, the solution was first dialyzed against large volumes of 0.075 M sodium chloride, in order to get its absolute concentration by dry weight. This salt level (one half the usual saline concentration) was chosen as one of several precautions to ensure an adequate degree of accuracy in the measurement. Since the protein concentration was expected to be rather low, it was important to have as small a salt correction as possible, but dialysis against salt-free water led to precipitation. Three samples, each of 1.00 ml., of the dialyzed solution were put into prepared weighing bottles and dried at 105O C. for 24 hours. At the same time, three samples, each of 10.00 ml., of the dialysis bath were similarly dried. After appropriate averaging and subtraction, a concentrat.ion of 0.050 per cent was determined for the macromolecular material.
0.030’
Fig.
Z.-Optical
density
plotted
against
’ 240
wave
’ ’ 260 Wavelength length
’
’ 280 Cm?) for
’
300
a preparation
of
Caddis
fly
PO01
2.
Another portion of this same stock solution was examined spectrophotometrically to obtain the extinction coefficient, and a third portion was placed directly into digestion tubes for the nitrogen determination. For the extinction study, a dilution series was made and the optical density (OD) was read at 280 rnp for each sample. A linear relation was obtained on plotting OD against c (the concentration, in per cent). The calculated extinction coefficient (E) was found to be 35.2, according to the equation, E = F+ This relationship is illustrated in Fig. 3. An additional study was made, using the biuret reaction, in the manner described previous1y.l Each of the dilutions was treated and measured in the spectrophotometer at 540 rnp. To correct for pigment color, each dilution was also measured without having been reacted with biuret reagent. Both sets of points give straight lines, as do also the respective differences, when plotted
4
SHULMAN,
BRONSON,
ARBESMAN
January-February,
J. Allergy 1963
against concentration. This is shown in Fig. 4. From the plot of differences, an extinction coefficient of 1.3 is calculated. For the nitrogen determination, three portions, 1.00 ml. each, of the same dialyzed stock solution were placed into three test tubes for Kjeldahl digestion. The final distilled products were titrated to give 0.0651, 0.0653, and 0.0640 mg. N per milliliter. Since the macromolecular concentration of this solution was 0.50 mg. per milliliter, the calculated nitrogen content is 13.0, 13.1, and 12.8 0.200
0.160 2
z ‘o
0.120
z-. 5 jj
0.080
E .u x O
0.040
0000” Fig.
3.-Optical
density
at
280
m&
’ ’ 0.10
’
030 Concentration
plotted against preparation.
Concentration
Fig. preparation the lower
’
’
040 (“/.x100)
’
concentration
0.60
of
a
Caddis
fly
Pool
2
(“6 x100)
4.-Optical density at 540 mc plotted against concentration of a Caddis fly Pool in the biuret reaction. The upper Zinc represents the reaction with biuret reagent; Z&e, with saline: the middle Ziae, the differences in the pairs of readings.
2
IMMUNOLOGIC
STUDIES
OF
CADDIS
FT,T.
III
5
per cent, respectively, for an average value of 13.0 per cent. As an additional precaution with regard to the experimental accuracy, the pipet delivering these samples wa.s subsequently calibrated especially for this experiment. The dclivered mass of water at 2~5.0~ C. from this pipet was found to be 0.9976 c&n. (average of three samples). Correcting for the density. the volume is calcnlatrtl to be 1.000 ml. Hesose Content.-Duplicate determinations werr made on samples of 0.50 ml. They agreed closely, and indicated a hexose concentration in the solution of 5.0 x IO-” (Im. per milliliter. Since the macromolecular material is itself 5.0 x 10m4Gm. per milliliter, the hexose content of it is one per cent. Ultracentrifugal Analysis.-The remaining quantity of this preparation was reconcentrated, and then dialyzed against 0.15 M NaCl. A sample at :I concentration of 0.48 per cent was studied in the ultracentrifuge at about 60,000 r.p.m., using a synthetic-boundary cell. The schlieren pattern showed a single.
Fig. S.--Sedimentation pattern of a Caddis fly Pool 2 preparation. Concentration of protein: 0.48 per cent. Photographs, left to right, at 0, 28, and 52 minutes after rotor reachecl speed of 58,900 r.p.m. Schlieren angles were 50, 40, 46 degrees, respectively. Direction of sedimentation in each picture is to the right.
protein: Time: turbidity
Fig.
6.-Electrophoretic pattern of a Caddis fly Pool 2 preparation. Concentration of 0.18 per cent. Barbital buffer: pH 8.60, I’/2 = 0.10. Electric field strength: 7.0 volt/cm. 60 min. Descending Pattern. The leading edge of the faster peak is obscured by the of the material.
6
SHULMAN,
BRONSON,
ARBESMAN
January-February,
J. Allergy 1963
symmetrical peak, showing no evident heterogeneity. Several pictures during the run are shown in Fig. 5. The sedimentation coefficient was calculated and corrected to the value in water at 20° C. This was 0.72 Svedberg unit. Electrophoretic Andy&s.-A portion of the preparation was dialyzed against the barbital buffer, pH 8.6, ionic strength 0.10. This was examined by optical electrophoresis. Because of the light brown color of the solution, it was not possible to obtain a complete pattern for either channel. A picture is given in Fig. 6. It can be seen that there are two components, of approximately equal amounts. The computed mobilities are -7.7 and -6.1 x lo+ cm.’ sec.-l volt-l. DISCUSSION
The data that have been accumulated on the Pool 2 preparation, previously shown to have high allergenic activity, are in agreement that the nature of this material is protein and that it has a rather low molecular weight. It may, therefore, be described as a polypeptide. The evidence can be summarized as follows. The ultraviolet absorption spectrum for Pool 2 is much more characteristic for protein, uncontaminated by nucleic acid, than is the spectrum for the original extract. In the extract, the ratio of optical densities at 2#60 and 280 my is 0.97, whereas purified serum albumin as a typical protein shows a ratio5 of 0.6, whereas that of nucleic acid is about 1.9. In the Pool 2 fraction, this ratio is reduced to 0.88. There may well be some residual nucleic acid, although a part of the increment from 0.6 may represent individual protein differences. The nitrogen content of 13.0 per cent indicates a composition close to that of a typical protein of 15 to 17 per cent nitrogen. A small amount of contaminating nucleic acid or polysaccharide may be thus indicated. The extinction coefficient at 280 rnp, 35.2, is somewhat high, but not unreasonable. It would indicate that there is a high content of tryptophan and/or tyrosine residues. The fact that the extinction coefficient of the initial extract’ was 14.1, a much lower figure, is not really incompatible with the value of 35.2, since the fraction represents only about 2 per cent of the total initial macromolecular material.3 The hexose determination eliminates the possibility that the active principle is a polysaccharide. The quantity of hexose is too small to seem significant by itself. While it is true that other types of sugar may in fact give some reaction in this calorimetric test, especially if present in favorable proportions to the hexose, the only important conclusion is that the amount of any carbohydrate material is limited to a very small percentage of the total high-molecular weight material. It remains quite possible that the allergenic protein is a glycoprotein or glycopeptide with as much as 1 per cent carbohydrate. Even if all the carbohydrate is in one of the components and assuming that only that one is the allergen, the carbohydrate content of the allergen would be, at most, 2 per cent. The ultracentrifugal examination reveals a single well-defined boundary, with a sedimentation coefficient which agrees well with that of the principal
Volume 34 Number I
IMMUNOLOGIC
STUDIES
OF
CADDIS
FLY.
I
III
component seen in the initial extra&l This was the slower of the two boundaries detected in the mixture. The present findings confirm our previous inference that the allergen itself was associated with this slow-sedimenting constituent. While a precise value of the molecular weight cannot be determined without additional data, a very reasonable value can be approximated by assuming the particle to be spherical and unhydrated. These assumptions, especially the first one, are quite reasonable for molecules that are clearly very One can then small compared to the usual range of protein molecular weights. apply the following formula (see, for example, Ref. 6) M = v/62 x2$ N?v [s,/(l - v)p]” where 31 is the molecular weight, s is the corrcctccl sedimentation coefficient, 7 is the relative viscosity of the solvent, N is the Avogadro number? < is the partial specific volume of the protein (assumed to be 0.75)) and p is the density of the solution. This calculation gives Bl = 3012, and therefore a molecular weight of 3000 can be taken as a reasonable estimate.
1. A purified fraction of Caddis Ay extract has been shown to consist of two components, which can be separated in electrophoresis, but not in the ultracentrifuge. The electrophoretic mobilities are -7.7 and -6.1 x lo-” cm.” sec.-’ volt-‘. The sedimentation coefficient is 0.72 S. 2. The estimated molecular weight for each component is 3000, assuming the particles to be spherical and unhydrated. 3. Extinction coefficients were determined for this preparation and found to be 35.2 by ultraviolet (280 rnp) and 1.3 by biuret (540 mp). 4. From the nitrogen content, spectral properties, hexose determination, and other data, it is concluded that the allergen is a peptide or glycopeptide. The authors determinations.
wish
to acknowledge
the
capable
assistance
of Joanna
Ferber
in the nitrogen
REFERESCES 1. Rapp, 2. 3. 4. 5. 6.
D., Shulman, S., and Arbesman, C. E.: immunologic Studies of Caddis Fly. 1. Preparation and Characterization of Extracts, J. ALLERGY 33: 97! 1962. Shulman, S., and Witebsky, E.: Studies on Organ Specificity. TX. Blophysical and Trnmunochemical Studies on Human Thyroid Autoantibody, J. Immunol. 85: 559, 1960. Shulman, S., Rapp, D.! Bronson, P., and Arbesman, C. E.: Immunologic Studies of Cadtlis Fly. II. Tsolatron of the Allergenic Fractions of Caddis Fly Extract, J. AJ,I,ERGY 33: 438, 1962. Winzler, R. J.: Determination of Serum Glycoproteins, Meth. Biochem. Bnal. 2: 279. 1955. Rhulman, S., and Bronson, P.: Unpublished experiments. Oncley, J. L.: Evidence From Physical Chemistry Regarding the Size and Shape of Protein Molecules From Ultra-eentrifugation, Diffusion, Viscosity, Dielectric Dispersion, and Double Refraction of Flow, Ann. New York Acad. SC. 41: 127, 1941.
Physical
Sidney
NO.
1
JANUARY-FEBRUARY,
1963
ARTICLES
IMMUNOLOGIC III.
v 0 I‘ . 34,
STUDIES
and Chemical
Shulman,
Ph.D.,*
Paul
OF CADDIS
Characterization Bronson,
and
FLY
of the Major Carl
E.
Arbesman,
Antigen &l.D.,
Buffalo,
N.
Y
of Caddis fly bodies, having potent allergenic activity, have been prepared. These have been fractionated by zone electrophoresis to yield >I selected product, termed Pool 2, containing a considerably higher specific allcrgenicit,y than any of the other fractions. This material has now been collected from repeated electrophoretic separations, and these have been combined in order to give enough material for chemical and physical characterization. These properties are reported here.
E
XTRACTS
MATERIALS
AND
METHODS
Caddis fly extract was prepared by the direct method, as described in the first paper of this series.l Electrophoretic separation was then carried out on these extracts by means of curtain electrophoresis, using the procedures previously described.“, 3 A volume of 10 ml. was applied for each run. The resulting sets of 32 tubes were examined for protein concentration, and the tubes within the group corresponding to Pool 2 were pooled and saved. All other fractions were discarded. For the studies reported here, it was essential to have the material considerably more concentrated than had been necessary for evaluation of the allergenicity. It was found very useful to use the method of dry sucroseosmosis for this purpose. The sample was placed in a tied dialysis bag (Visking From the Departments of Bacteriology and Immunology and of Medicine, University of Buffalo School of Medicine, and the Allergy Research Laboratory of the BufPalo General Hospital, Buffalo. N. Y. This investigation was supported in part by Research Grants, E-1303 from the National Institute of Allergy and Infectious Diseases, and A-4195 from the National Institute of Arthritis and Metabolic Diseases, U. S. Public Health Service, Bethesda, Md. Received for publication July 30, 1962. *Recipient of a Research Career Development Awar (GM-K3-1377) from the U. S. Public Health Service.
2
SHULMAN,
BBONSON,
ARBESMAN
January-February,
J. Allergy 1963
cellophane) and put in a glass dish. Dry sucrose was poured on and around it. After several hours, the volume of solution in the bag had decreased to about one half, or less. The wet sucrose was washed away and the bag was tied again at the smaller volume. It could then be dialyzed against a saline or buffer solution, as desired. Analyses by ultracentrifuge and by optical electrophoresis were done and computed by standard methods, using the Spinco Models E and H, respectively. Nitrogen analysis was conducted by a semi-micro Kjeldahl method. Titration was performed with 0.01 N HCl, using methyl purple as an indicator. Light absorption studies were made with a Beckman DU spectrophotometer. Hexose was determined by the orcinol method, using a galactose-mannose standard, as described by Winzler.4 1.200
1.000
-
a c z 0
0.000
-
;
0.600
-
0.400
-
0.200
-
u Ti 0
0.000
Fig.
1.4ptical
density
plotted
’ 240
against
’
’ 260 Wavelength wave
’
’ 280 (mp)
length
’
for
: 10
a direct
Caddis
fly
extract.
RESULTS
Ultraviolet Spectrum.-A sample of the original Caddis fly extract was examined for its light absorption over the range of wave lengths from 220 to 290 rnp. The spectrum is shown in Fig. 1. The anticipated maximum for protein (at about 280 mp) is poorly resolved on the low wave length side, presumably because of the presence of an appreciable amount of nucleic acids and polynucleotides, absorbing maximally at 260 mp. The protein absorption seems to be maximal at about 275 mp. A spectrum of t.his type for the Pool 2 preparation is shown in Fig. 2. It is somewhat similar to that for the whole extract, although a better degree of resolution at 280 rnp is now evident; the polynucleotide absorption is proportionately decreased. Extinction Coefficients and Nitrogen Content,.-To explore the question of whether or not this purified substance is a protein, or whether perhaps partly
Volume 34 Number I
IMMUNOLOGIC
STUDIES
OF
(IADDIS
FL‘T-.
III
3
protein, a nitrogen analysis was carried out. To make this information meaningful, the solution was first dialyzed against large volumes of 0.075 M sodium chloride, in order to get its absolute concentration by dry weight. This salt level (one half the usual saline concentration) was chosen as one of several precautions to ensure an adequate degree of accuracy in the measurement. Since the protein concentration was expected to be rather low, it was important to have as small a salt correction as possible, but dialysis against salt-free water led to precipitation. Three samples, each of 1.00 ml., of the dialyzed solution were put into prepared weighing bottles and dried at 105O C. for 24 hours. At the same time, three samples, each of 10.00 ml., of the dialysis bath were similarly dried. After appropriate averaging and subtraction, a concentrat.ion of 0.050 per cent was determined for the macromolecular material.
0.030’
Fig.
Z.-Optical
density
plotted
against
’ 240
wave
’ ’ 260 Wavelength length
’
’ 280 Cm?) for
’
300
a preparation
of
Caddis
fly
PO01
2.
Another portion of this same stock solution was examined spectrophotometrically to obtain the extinction coefficient, and a third portion was placed directly into digestion tubes for the nitrogen determination. For the extinction study, a dilution series was made and the optical density (OD) was read at 280 rnp for each sample. A linear relation was obtained on plotting OD against c (the concentration, in per cent). The calculated extinction coefficient (E) was found to be 35.2, according to the equation, E = F+ This relationship is illustrated in Fig. 3. An additional study was made, using the biuret reaction, in the manner described previous1y.l Each of the dilutions was treated and measured in the spectrophotometer at 540 rnp. To correct for pigment color, each dilution was also measured without having been reacted with biuret reagent. Both sets of points give straight lines, as do also the respective differences, when plotted
4
SHULMAN,
BRONSON,
ARBESMAN
January-February,
J. Allergy 1963
against concentration. This is shown in Fig. 4. From the plot of differences, an extinction coefficient of 1.3 is calculated. For the nitrogen determination, three portions, 1.00 ml. each, of the same dialyzed stock solution were placed into three test tubes for Kjeldahl digestion. The final distilled products were titrated to give 0.0651, 0.0653, and 0.0640 mg. N per milliliter. Since the macromolecular concentration of this solution was 0.50 mg. per milliliter, the calculated nitrogen content is 13.0, 13.1, and 12.8 0.200
0.160 2
z ‘o
0.120
z-. 5 jj
0.080
E .u x O
0.040
0000” Fig.
3.-Optical
density
at
280
m&
’ ’ 0.10
’
030 Concentration
plotted against preparation.
Concentration
Fig. preparation the lower
’
’
040 (“/.x100)
’
concentration
0.60
of
a
Caddis
fly
Pool
2
(“6 x100)
4.-Optical density at 540 mc plotted against concentration of a Caddis fly Pool in the biuret reaction. The upper Zinc represents the reaction with biuret reagent; Z&e, with saline: the middle Ziae, the differences in the pairs of readings.
2
IMMUNOLOGIC
STUDIES
OF
CADDIS
FT,T.
III
5
per cent, respectively, for an average value of 13.0 per cent. As an additional precaution with regard to the experimental accuracy, the pipet delivering these samples wa.s subsequently calibrated especially for this experiment. The dclivered mass of water at 2~5.0~ C. from this pipet was found to be 0.9976 c&n. (average of three samples). Correcting for the density. the volume is calcnlatrtl to be 1.000 ml. Hesose Content.-Duplicate determinations werr made on samples of 0.50 ml. They agreed closely, and indicated a hexose concentration in the solution of 5.0 x IO-” (Im. per milliliter. Since the macromolecular material is itself 5.0 x 10m4Gm. per milliliter, the hexose content of it is one per cent. Ultracentrifugal Analysis.-The remaining quantity of this preparation was reconcentrated, and then dialyzed against 0.15 M NaCl. A sample at :I concentration of 0.48 per cent was studied in the ultracentrifuge at about 60,000 r.p.m., using a synthetic-boundary cell. The schlieren pattern showed a single.
Fig. S.--Sedimentation pattern of a Caddis fly Pool 2 preparation. Concentration of protein: 0.48 per cent. Photographs, left to right, at 0, 28, and 52 minutes after rotor reachecl speed of 58,900 r.p.m. Schlieren angles were 50, 40, 46 degrees, respectively. Direction of sedimentation in each picture is to the right.
protein: Time: turbidity
Fig.
6.-Electrophoretic pattern of a Caddis fly Pool 2 preparation. Concentration of 0.18 per cent. Barbital buffer: pH 8.60, I’/2 = 0.10. Electric field strength: 7.0 volt/cm. 60 min. Descending Pattern. The leading edge of the faster peak is obscured by the of the material.
6
SHULMAN,
BRONSON,
ARBESMAN
January-February,
J. Allergy 1963
symmetrical peak, showing no evident heterogeneity. Several pictures during the run are shown in Fig. 5. The sedimentation coefficient was calculated and corrected to the value in water at 20° C. This was 0.72 Svedberg unit. Electrophoretic Andy&s.-A portion of the preparation was dialyzed against the barbital buffer, pH 8.6, ionic strength 0.10. This was examined by optical electrophoresis. Because of the light brown color of the solution, it was not possible to obtain a complete pattern for either channel. A picture is given in Fig. 6. It can be seen that there are two components, of approximately equal amounts. The computed mobilities are -7.7 and -6.1 x lo+ cm.’ sec.-l volt-l. DISCUSSION
The data that have been accumulated on the Pool 2 preparation, previously shown to have high allergenic activity, are in agreement that the nature of this material is protein and that it has a rather low molecular weight. It may, therefore, be described as a polypeptide. The evidence can be summarized as follows. The ultraviolet absorption spectrum for Pool 2 is much more characteristic for protein, uncontaminated by nucleic acid, than is the spectrum for the original extract. In the extract, the ratio of optical densities at 2#60 and 280 my is 0.97, whereas purified serum albumin as a typical protein shows a ratio5 of 0.6, whereas that of nucleic acid is about 1.9. In the Pool 2 fraction, this ratio is reduced to 0.88. There may well be some residual nucleic acid, although a part of the increment from 0.6 may represent individual protein differences. The nitrogen content of 13.0 per cent indicates a composition close to that of a typical protein of 15 to 17 per cent nitrogen. A small amount of contaminating nucleic acid or polysaccharide may be thus indicated. The extinction coefficient at 280 rnp, 35.2, is somewhat high, but not unreasonable. It would indicate that there is a high content of tryptophan and/or tyrosine residues. The fact that the extinction coefficient of the initial extract’ was 14.1, a much lower figure, is not really incompatible with the value of 35.2, since the fraction represents only about 2 per cent of the total initial macromolecular material.3 The hexose determination eliminates the possibility that the active principle is a polysaccharide. The quantity of hexose is too small to seem significant by itself. While it is true that other types of sugar may in fact give some reaction in this calorimetric test, especially if present in favorable proportions to the hexose, the only important conclusion is that the amount of any carbohydrate material is limited to a very small percentage of the total high-molecular weight material. It remains quite possible that the allergenic protein is a glycoprotein or glycopeptide with as much as 1 per cent carbohydrate. Even if all the carbohydrate is in one of the components and assuming that only that one is the allergen, the carbohydrate content of the allergen would be, at most, 2 per cent. The ultracentrifugal examination reveals a single well-defined boundary, with a sedimentation coefficient which agrees well with that of the principal
Volume 34 Number I
IMMUNOLOGIC
STUDIES
OF
CADDIS
FLY.
I
III
component seen in the initial extra&l This was the slower of the two boundaries detected in the mixture. The present findings confirm our previous inference that the allergen itself was associated with this slow-sedimenting constituent. While a precise value of the molecular weight cannot be determined without additional data, a very reasonable value can be approximated by assuming the particle to be spherical and unhydrated. These assumptions, especially the first one, are quite reasonable for molecules that are clearly very One can then small compared to the usual range of protein molecular weights. apply the following formula (see, for example, Ref. 6) M = v/62 x2$ N?v [s,/(l - v)p]” where 31 is the molecular weight, s is the corrcctccl sedimentation coefficient, 7 is the relative viscosity of the solvent, N is the Avogadro number? < is the partial specific volume of the protein (assumed to be 0.75)) and p is the density of the solution. This calculation gives Bl = 3012, and therefore a molecular weight of 3000 can be taken as a reasonable estimate.
1. A purified fraction of Caddis Ay extract has been shown to consist of two components, which can be separated in electrophoresis, but not in the ultracentrifuge. The electrophoretic mobilities are -7.7 and -6.1 x lo-” cm.” sec.-’ volt-‘. The sedimentation coefficient is 0.72 S. 2. The estimated molecular weight for each component is 3000, assuming the particles to be spherical and unhydrated. 3. Extinction coefficients were determined for this preparation and found to be 35.2 by ultraviolet (280 rnp) and 1.3 by biuret (540 mp). 4. From the nitrogen content, spectral properties, hexose determination, and other data, it is concluded that the allergen is a peptide or glycopeptide. The authors determinations.
wish
to acknowledge
the
capable
assistance
of Joanna
Ferber
in the nitrogen
REFERESCES 1. Rapp, 2. 3. 4. 5. 6.
D., Shulman, S., and Arbesman, C. E.: immunologic Studies of Caddis Fly. 1. Preparation and Characterization of Extracts, J. ALLERGY 33: 97! 1962. Shulman, S., and Witebsky, E.: Studies on Organ Specificity. TX. Blophysical and Trnmunochemical Studies on Human Thyroid Autoantibody, J. Immunol. 85: 559, 1960. Shulman, S., Rapp, D.! Bronson, P., and Arbesman, C. E.: Immunologic Studies of Cadtlis Fly. II. Tsolatron of the Allergenic Fractions of Caddis Fly Extract, J. AJ,I,ERGY 33: 438, 1962. Winzler, R. J.: Determination of Serum Glycoproteins, Meth. Biochem. Bnal. 2: 279. 1955. Rhulman, S., and Bronson, P.: Unpublished experiments. Oncley, J. L.: Evidence From Physical Chemistry Regarding the Size and Shape of Protein Molecules From Ultra-eentrifugation, Diffusion, Viscosity, Dielectric Dispersion, and Double Refraction of Flow, Ann. New York Acad. SC. 41: 127, 1941.