Int. Archs Allergy appl. Immun.

65: 367-376 (1981)

Isolation of Allergenically Active Cytochrome c from Kentucky

Blue Grass Pollen1
A. K. M. Ekramoddoullah, F. T. Kisil, A. H. Selion12
MRC Group for Allergy Research, Department of Immunology, Faculty of Medicine,
University of Manitoba, Winnipeg, Man., Canada

Abstract. An allergen, designated as Kentucky blue grass KBG-cytochrome c, was

isolated from the nondialysable constituents (R) of the aqueous extract of KBG pollen by
a combination of gel filtration and preparative isoelectrofocussing techniques. The allergen
had an absorption spectrum characteristic of heme protein and had a molecular weight
corresponding to 12,000 daltons. It was a basic protein with a pi value of 9.9 and contained
all the common amino acids. KBG-cytochrome c elicited immediate hypersensitivity
cutaneous reactions in individuals allergic to KBG pollen.

Introduction quencc, they all have an identical 3-dimcn-

sional structure. Thus, cytochromes c repre­
Cytochrome c has been extensively used sent an excellent group of proteins for study­
as a prime model for establishing the evolu­ ing the effects of differences in amino acid
tionary developments leading to the diver­ sequence on the antigenicity of a globular
gence of eukaryotes. The phylogenetic rela­ protein with unvarying conformation. Sev­
tionships of these organisms have been ob­ eral investigators [3, 16, 23-27] have used
tained from the comparative analysis of the cytochrome c as a model antigen for localiz­
amino acid sequence data of cytochromes c. ing antigenic determinants within the mole­
As a result of this interest in the evolution­ cule and also for dissecting the cellular im­
ary history, the primary structures of 85 cy­ mune response to a globular protein. These
tochromes c of different origin have been studies were accomplished by relating the
elucidated [4], Although cytochromes c of extent of the immune cross-reactivity at
diverse origin, e.g. horse, bonito and tuna, both the humoral and cellular levels, of var­
possess a wide variation in amino acid se- ious cytochromes c to differences in their
amino acid sequences.
1 This work was supported by grants from the
The recent finding of Goodjriend et al.
Medical Research Council of Canada and the Na­
tional Institutes of Health (AI 14526), Bcthesda, Md. [14] that cytochrome c isolated from rag­
2 The capable technical assistance of Tom Cook weed pollen was allergenic in ragweed-sen­
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is gratefully acknowledged. sitive individuals suggests the possibility

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368 Ekramoddoullah/KisilSehon

that cytochromes c present in other pollens Ont., Canada) against distilled water for 48 h at
may also have allergenic activity. If other 4 C; the nondialyzable components are referred
pollen cytochromes c are indeed found to be to as the reténtate (R).
allergenic, it would be of interest to deter­ Chromatography
mine the extent, if indeed any, of their aller­ Sephadex G-50 (particle size 20-80 //in; Phar­
genic cross-reactivity. macia AB. Uppsala, Sweden) was equilibrated with
0.05 M Tris-HCl, pH 8.6, containing 0.02°/o NaNs
Because the structural features of differ­ and packed into a column (2.5 X 90 cm) at 4 °C.
ent cytochromes c have been highly con­ Ultrogel ACA-44 (LKB 2204-440, Industrie
served during evolutionary changes, pollen Biologique Française, Genevilliers, France) was
cytochromes from related species within a equilibrated with 0.05 M phosphate buffer, pH
family, e.g. Graminae (grass), may be ex­ 7.2, containing 0.15 M NazSO/, 0.02°/o NaNa and
packed into a column (2.5 X 90 cm).
pected to differ minimally in their amino All chromatographic eluates were concentrated
acid sequences. These minor differences by ultrafiltration through UM-2 membrane, with
may help to identify specific regions of the a cutoff for molecules larger than 1.000 daltons
cytochrome molecule which are responsible (Amicon, Lexington, Mass.). Desalting of the con­
for the allergenic activities. This approach centrated eluates was achieved by dialysis through
Spectrapor membrane tubing (specified molecular
would be similar to that which has been suc­ weight cutoff = 3,500; Spectrum Medical Indus­
cessfully used to identify three regions with­ tries, Inc., Los Angeles, Calif.) for 24 h against a
in the primary structure that have been im­ large volume of distilled water with three changes.
plicated in the antigenicity of mammalian
Preparative Isoelectrofocussing (ISO-EF)
cytochromes c (e.g. human, horse, guanaco The procedure of ISO-EF recently described
and mouse) [23], [II] was adapted with the following modifications.
In earlier studies from our laboratory, Thus, 5.5 g of Ultrodex (LKB-Productor AB,
protocols were developed for isolating puri­ Stockholm-Bromal, Sweden) was swollen in
142.5 ml of a solution of fraction R-I (see Results,
fied allergens from the aqueous extract of
p. 370) at a concentration of 4 mg/ml water. A vol­
KBG pollen [1, 2, 10-12]. In the present ume of 7.5 ml of 400/o ampholyte buffer, pH range
study, we report the isolation of an allergen- 3.5-10 (LKB), was added to the gel suspension,
ically active cytochrome c from the nondia- which was then deaerated under vacuum. The gel
lysable multicomponent fraction, i.e. retén­ was poured onto a horizontal glass tray (11 X 30
X 0.8 cm, Bio-Rad Laboratories, Richmond, Cal­
tate (R), of the aqueous extract of Kentucky
if.), spread uniformly and dried to a weight loss of
blue grass (KBG) pollen by a combination about 30'7o. Three strips of paper wicks (0.7 X
of gel filtration and isoelectrofocussing tech­ 11cm, Bio-Rad) presoaked with 0.2 M H î SO< for
niques. the anode and another three strips presoaked with
0.4 M ethylenediamine for the cathode were then
laid on top of the gel at the far ends; ISO-EF elec­
Materials and Methods trodes, 110 mm (Bio-Rad) were placed on top of
the wicks and the ISO-EF was carried out as de­
Preparation of R scribed previously [11],
An aqueous extract of KBG pollen (Hollister- After ISO-EF, the gel was divided into 38
Stier Laboratory, Mississauga, Ont., Canada) was equal sections with the aid of a gel divider (Bio-
prepared according to the procedure previously Rad) and the pH of each gel section was measured
established in this laboratory [7]. The aqueous ex­ with the aid of surface pH electrode (Bio-Rad).
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tract was dialyzed through visking tubing, No. 20 Each gel section was poured into a PEGG elution
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dialysis (Union Carbide Canada Ltd., Lindsay, column (LKB) and eluted with 5 ml of water. The
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Cytochrome c of Kentucky Blue Grass Pollen 369

absorbance of the eluates at 280 nm was measured diamine (TEMED) to the 10 ml of gel mixture and
in a Perkin-Elmer UV-VIS spectrophotometer poured into glass tubes (0.5 X 12.5 cm). The stack­
(Coleman 139, Coleman Instrument Division, ing gel was prepared in the same way, except that
Maywood, III,). All fractions were dialyzed exten­ the concentration of acrylamide and methyl bis-
sively against water through Spectrapor membrane acrylamide was 3 and 0.8%>, respectively, in
tubing (molecular cutoff 6,000-8,000) and lyo- 0.125 M Tris-HCl, pH 6.8. The electrode buffer
philized. (pH 8.3) contained 0.025 M Tris and 0.192 M
glycine ar.d 0.1'Vo SDS. The sample buffer consisted
Protein Measurement of 0.0625 M Tris-HCl (pH 6.8), 2°/o SDS, 10%
The protein content of pollen fractions was de­ glycerol, 5% 2-mercaptoethanol, 0.001°/« bromo-
termined by the method of Lowry et al. [21 ]. Un­ phenol blue. The sample was dissolved in sample
less stated otherwise, amounts or concentrations buffer and boiled for 90 s. Electrophoresis was
of various fractions are expressed in terms of their carried out with a current of 3 niA/tubc until the
protein content. bromophenol blue marker reached the bottom of
the gel. The gels were stained according to the
Absorption Spectrum method of Fairbanks et al. [13], The following
The sample was dissolved in water and read at proteins with the indicated molecular weights
wavelengths of 5-nm intervals in a Zeiss spectro­ were used as markers: human serum albumin,
photometer (Zeiss, Obcrkochen, FRG). 68,000 (Pentex, Miles Laboratories, Elkhart, lnd.);
ovalbumin, 43,000 (1CN Pharmaceuticals, Cleve­
Chemical Analysis
land, Ohio); chymotrypsinogen A, 25.700, ribonu-
The amino acid analysis was kindly performed
clease A, 13,700 (Worthington Biochemical Corp.,
by Dr. F. Stevens of the Department of Biochem­
Freehold, N.J.); pepsin, 35,000 trypsin, 23,300,
istry, University of Manitoba. Samples were hy­
horse heart cytochrome c, 11,700 (Sigma, St.
drolyzed with HC1 at 110-112 C for 24, 48 and
Louis, Md.). The mobility of the separated protein
72 h. The hydrolysates were analyzed with the
was measured as described by Weber and Osborn
Beckman automatic amino acid analyzer, Model
[28],
120-139 (Beckman, Palo Alto, Calif.). The con­
Alternatively, the molecular weights of pollen
centration of serine, theonine and tyrosine was
fractions were estimated by gel filtration through
calculated by extrapolation to zero time hydrolysis
Ultrogel ACA-44 calibrated with proteins listed
and that of valine and isoleucine was calculated
above.
from the analysis of 72 h hydrolysate. The cys­
teine was determined as cysteic acid by oxidizing
the sample with performic acid [15] prior to 24 h Radioallergosorbent Test (RAST)
acid hydrolysis. The tryptophan content was mea­ The test recently described [II] was used with
sured spectrophotometrically [6], The concentra­ the following modifications. CNBr-activated paper
tion of other amino acids was calculated from the discs (one disc/100 «g of sample to be coupled)
analysis of the 48 h hydrolysate. Qualitative tests were suspended in a solution of a pollen fraction
for carbohydrate were performed by a spot test as (1.0 mg/ml of 0.1 M NaHCOs) for 18 h at 4 °C.
described elsewhere [11]. Unreacted materials were removed by washing the
paper discs twice with 0.1 M NaHCOs. To block
Molecular Weight Estimation any residual active sites on the cellulose, the discs
The molecular weight of pollen constituents were reacted with a solution of /J-ethanolamine
was determined by electrophoresis in polyacrylam­ (0.5 M in 0.1 M NaHCOi) for 3h and then
ide gel in presence of sodium dodecyl sulfate washed with 0.1 M sodium acetate buffer, pH 4.0,
(SDS-PAGE) as described by Laemmli [18], The followed by extensive washing with phosphate-
separation gel consisted of 10 g acrylamide, 0.8 g buffered saline (7.75 g NaCI, 0.54 g KH2 PCL,
methyl bis-acrylamide, 1 g SDS, 0.4 g ammonium 2.14 g NaiHPOi in liter of H2 O, pH 7.2; PBS).
persulfate dissolved in total volume of 100 ml of The discs were stored in incubation buffer (PBS
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0.375 M Tris-HC'l, pH 8.8. The gel was polymer­ containing 0.3°/« bovine serum albumin and 0.02%
ized by adding 0.025 ml of tetramethylethylene- NaNj).
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370 Ekramoddoullah/Kisil/Sehon

Volumes of 0.05 ml of a pool of human sera

from 6 patients allergic to KBG were incubated
overnight at 4 C with the allergosorbent discs
and then washed with incubation buffer. The ex­
tent to which the IgF. antibodies bound to the in-
solubilized allergens was evaluated by the addition
of ,25I-labclled rabbit anti-human IgE antibodies
bound to the discs was measured with a Beckman
Gamma 300 System counter.
For the experiments involving the inhibition of
RAST, 0.05 ml volume of allergic sera was mixed
with different amounts of each pollen fraction and
maintained for 3 h at room temperature and then
added to the allergosorbent discs. Fig. 1. Gel filtration of R on Sephadex G-50
(particle size, 20-80/tm). Column size 2.5 X
Skin Tests 90 cm; eluting buffer: 0.05 M Tris-HCI, pH 8.6,
The skin tests were kindly performed by Dr. containing 0.02“/o NaNs.
ii. Warrington of this Department. A stock solu­
tion of 0.1 mg/ml of fraction in saline was steri­
lized by passing through a sterile Millipore mem­ and R-II (fig. 1). The chromatography also
brane (0.45 ttm pore size). A 20-«l volume of a
resulted in the removal of some low molecu­
100-fold diluted stock solution was injected in-
tradermally into patients sensitive to KBG. 15 min lar weight yellow-pigmented materials,
later, the size of the wheal was measured. which were retarded in the column and,
therefore, were eluted much later than R-II.
Passive Cutaneous Anaphylaxis (PCA) It was found that the absorption of fraction
This procedure was employed to establish the
R-I at 410 nm relative to its absorption at
PCA activity of isolated fractions relative to that of
R. For this purpose, IgE antibodies to the aller­ 280 nm was higher than that of R, whereas
gens present in R were produced in mice and were R-II contained very little material which ab­
determined in terms of their PCA titer on the skin sorbed at 410 nm. Since the absorption at
of rats, as previously described (8, 9]. For the ac­ 410 nm is one of the characteristic absorp­
tual determination of PCA activity, hooded rats
were locally sensitized by the intradermal injec­
tion maxima of cytochrome c, it would ap­
tion of a volume of 50 «1 of the mixture and 24 h pear that the gel filtration of R yielded a
later the PCA reaction, if any, was elicited by in- fraction, i.e. R-I, which was enriched in cy­
traveneous (i.v.) administration of 0.1 mg of a pol­ tochrome c content. Fraction R-I was there­
len fraction in Evan's blue dye (l»/o in saline); a fore rechromatographed on Sephadex G-50
PCA reaction was considered positive when the
and then subjected to preparative ISO-EF
extent of the extravasation of the dye, as mea­
sured on the underside of the skin, was 5 mm in on Ultrodex in the pH range of 3.5-10. The
diameter or greater. gel containing the focussed components was
subdivided into 38 fractions, including an­
ode and cathode fractions, i.e. the fractions
Results underneath the electrode wicks. As is shown
in figure 2, fraction R-l was resolved into
The reténtate fraction, R, was resolved several components. A distinct red-colored
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by chromatography on Sephadex G-50 into band was localized within the cathode frac­
two fractions arbitrarily designated as R-I tion.
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Cytochrome c of Kentucky Blue Grass Pollen 371

Allergenic Activity of Subfractions of R-l

The allergenic activity of these 38 sub­
fractions was evaluated by inhibition of the
RAST utilizing allergosorbent discs pre­
pared with R and a pool of human anti-
KBG reaginic sera. The results listed in
table 1 indicate that all 38 subfractions in­
hibited the RAST to varying degrees and,
therefore, indicated that they all possessed
Fig. 2. Preparative ISO-EF of R-I on Ultrodex allergenically active components.
(ampholine: pH 3.5-10; 96 h).

Isolation of Cytochrome c from Cathode

Fraction
An analysis of the absorption spectrum
Table I. Allergenic activity of focussed fractions
of R-l of the cathode fraction showed that it had
absorption maxima at 280, 410. 520 and
Fraction No. Inhibition Fraction No. Inhibition 550 nm and similar to that of cytochrome c.
of of SDS-PAGE analysis revealed that the cath­
RAST1, % RAST', % ode fraction contained three components
Anode (A) 70.0
with molecular weights corresponding to
19 59.0
1 68.5 20 65.4 53,000, 28,500 and 12,000 dallons. These
2 42.9 21 65.0 components were further resolved by chro­
3 51.8 22 71.5 matography on Ultrogel ACA 44 (fig. 3).
4 30.5 23 73.8 Thus, five fractions, designated as I, II, III,
5 33.0 24 72.3
6 34.0 25 64.0
7 40.5 26 77.4
8 33.7 27 72.8
9 29.5 28 72.4
10 23.4 29 74.7
II 25.3 30 64.7
12 34.0 31 58.0
13 42.6 32 59.7
14 46.5 33 57.0
15 49.8 34 55.0
16 55.0 35 57.6
17 57.9 36 52.6
18 59.3 cathode (C) 51.9
R 90.7
V O LU M E O F EFFLUENT, ml
1 100 pg (protein) of each fraction or of R was incu­
bated with a pool of sera from humans allergic to Fig. 3. Gel filtration of cathode fraction on
KBG pollen, prior to addition to the allergosorbent Ultrogel AC A 44. Column size 2.5 X 90 cm; elut­
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disc prepared with R. ing buffer: 0.05 M phosphate buffer, pH 7.2, con­
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taining 0.15 M NaaSOi and 0.02°/o NaN3.

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372 Ekramodoullah/Kisil/Sehon

IV and V contained material with high opti­

cal density profiles at 280 nm. However,
only fraction III showed high absorption at
410 nm, while fraction IV contained a small
amount of material which absorbed at
410 nm. Fraction III was rechromato­
graphed on a column of Ultrogel ACA 44
calibrated with proteins of known molecular
weight and it was found that the elution vol­
ume of fraction III corresponded to that of
horse cytochrome c. Fraction III will here­ W A V EL E N G T H (nm)

after be referred to as KBG-cytochrome c.

Fig. 4. Absorption spectrum of KBG-cyto-
chromc c (0.7 mg/ml HsO) at room temperature.
Immunological and Physicochemical
Properties of KBG-Cytochrome c
It was shown by the RAST procedure
that the allergosorbent disc prepared with
100//g of KBG-cytochrome c and incubat­
ed with 50 p\ of a pool of the allergic sera
was capable of binding 20°/o of 125I-antihu-
man IgE added, as compared to 37°/o of the
radioactivity bound by the allergosorbent
disc prepared with 100 pg of R. Further ev­
idence for the allergenicity of KBG-cyto­
chrome c was obtained by direct skin testing
of 3 KBG-sensitive patients (whose sera
were not included in the pool of sera used in
RAST). KBG-cytochrome c at a concentra­
tion of 1 //g/ml gave positive skin reactions
in 2 patients, the size of the wheal being 12
X 8 and 8 X 8 mm, respectively. The 3rd
patient did not react to this challenge. Con­
sistent with this finding, KBG-cytochrome c
was also demonstrated to be capable of elic­
iting PCA reactions in rats passively sensi­
i
tized with a murine anti-R reaginic sera.
The PCA titer of the murine reaginic sera
obtained by i.v. challenge of 100 //g of
KBG-cytochrome c was 160 (PCA titer elic­
ited with 100 iig of R was 640).
Fig. 5. Polyacrylamide gel electrophoresis of
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The absorption spectrum of KBG-cyto­ 50 ¡tg protein of KBG-cytochrome c in presence

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chrome c is shown in figure 4. Thus, of SDS.

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Cytochrome c of Kentucky Blue Grass Pollen 373

Table II. Amino acid composition of KBG-cy- band corresponding to a molecular weight
tochromec of the order of 12,000 daltons in
SDS-PAGE (fig. 5). The pi of KBG-cyto­
Amino acid Residue per molecule
chrome c, as determined by preparative
Alanine 9
ISO-EF of KBG-cytochrome c was 9.9. The
Arginine 4 amino acid analysis revealed that it con­
Aspartic acid 10 tained all the naturally occurring amino ac­
Cysteine 2 ids (tabic II). KBG-cytochrome c did not
Glutamic acid II contain carbohydrate, since it failed to give
Glycine 10
a positive test for carbohydrate.
Histidine 2
Ileucine 3
Leucine 8
Lysine 11 Discussion
Methionine 2
Phenylalanine 5
Proline 9
In this study allergenically active cyto­
Serine 5 chrome c was isolated from the nondialysa-
Threonine 7 ble fraction R of KBG pollen. The consti­
Tryptophan 2 tuents of R were first separated, on the basis
Tyrosine 4 of their size, into two fractions and the high­
Valine 8
er molecular weight fraction, R-I, which
was enriched with cytochrome c like materi­
al, was further resolved by preparative ISO-
EF into fractions with pi values ranging
KBG-cytochrome c had absorption maxima from 3.5 to 10.
at 280, 350, 410, 520 and 550 nm. While Although all the focussed fractions of
the absorption maxima at 280 nm is due to R-I contained allergenic components, only
protein, the maxima at 350, 410, 520 and the cathode fraction was enriched with red-
550 are the characteristics of the hemo- colored material and had an absorption
chrome spectrum of cytochrome c [22]. spectrum characteristic of cytochrome c.
KBG-cytochrome c is red in color; if, how­ SDS-PAGE analysis revealed that the cath­
ever, a few crystals of sodium dithionate ode fraction contained a component with a
were added to a solution of KBG-cyto­ molecular weight characteristic of cyto­
chrome c, the color changed to pink. Under chrome c and two additional components
this reduced conditions, the absorption with molecular weights higher than that of
maxima at 410 shifted to 415, the absorp­ cytochrome c. The cathode fraction was re­
tion maxima at 550 was more pronounced solved on ACA 44 into five fractions, all of
while the absorption maxima at 520 was which were capable of binding human IgE
barely detectable. These data would indicate antibodies to allergens of KBG pollen. The
that cytochrome c, isolated from KBG pol­ two low molecular weight fractions, i.e.
len, was predominantly in oxidized form. fractions IV and V, were easily dialysable
KBG-cytochrome c was shown to be through Spectrapor membrane tubing (mo­
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homogeneous, since it moved as a single lecular cut off = 6-8 X 103) and, conse­
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374 Ekramoddoullah/Kisil/Sehon

quently, were not detectable by SDS-PAGE Thus, KBG-cytochrome c did not precipi­
analysis. tate with rabbit antiserum prepared against
Fraction III (fig. 3) was identified as allergen C. Allergen C contained approxi­
KBG-cytochrome c by its color, absorption mately 30°/ci carbohydrate whereas no car­
spectrum, size, charge and amino acid com­ bohydrate was detected in KBG-cytochrome
position similar to those cytochromes isolat­ c. A complete analysis of antigenic and al­
ed from other plant sources [4], Al­ lergenic relationships of KBG-cytochrome c
though KBG-cytochrome c is a highly basic and allergen C must await the availability of
protein (pi value = 9.9), the acidic amino appropriate antisera to KBG-cytochrome c.
acids, i.e. aspartic acid and glutamic acid, The salient feature of cytochrome c iso­
were found to be present in amounts higher lated from KBG pollen was that it elicited
than the basic amino acids, i.e. arginine, his­ allergic reactions in individuals sensitive to
tidine and lysine. Since asparagine and glut­ KBG pollen. The fact that cytochrome c of
amine in a protein are converted into glut­ KBG and ragweed pollen were demonstrat­
amic and aspartic acids, respectively, dur­ ed to be allergenic in man strongly suggests
ing acid hydrolysis, the amounts of aspartic that the various pollens may contain cyto­
and glutamic acid determined by the acid chrome c as a part of their allergenic consti­
hydrolysis of KBG-cytochrome c may in­ tuents. As referred to in the Introduction,
deed be higher than their actual values. To the isolation of cytochromes c of various
obtain the true values, it will be necessary to grass pollens may represent a group of
identify the individual amino acids in the cross-reactive allergens. The availability of
course of the complete sequence analysis of well-characterized and closely-related aller­
KBG-cytochrome c. A striking difference gens is expected to pave the way for localiz­
noted between KBG-cytochrome c and two ing the allergenic sites on the molecule by
glycoprotein allergens, C-I-2-6 [11] and al­ correlation of allergenic activity with dis­
lergen C [2] also isolated from KBG pollen, tinct region(s) identified from amino acid
was that the cytochrome c allergen was de­ sequence analysis. Once the allergenic de­
void of carbohydrate. An analysis of aller­ terminants) are identified, it can be antici­
genic relationships between the two glyco­ pated that the synthesis of univalent deriva­
protein allergens and KBG-cytochrome c tives of the allergenic determinants may be
may aid in the understanding of the role of achieved.
carbohydrate (if any) on the allergenicity of The univalent determinants would serve
the two glycoprotein allergens. It should be as immunotherapeutic agents, since these
noted that the color, size and charge charac­ compounds on administration would inhibit
teristics of cytochrome c closely resembled the allergic reactions triggered by the cross-
that of allergen C [2] and one may therefore linking of cell-fixed IgE antibodies by multi­
contend that cytochrome c might be con­ valent allergens. For example, the treatment
taminated with allergen C. Although such a of penicillin allergy by the injection of large
possibility cannot be completely ruled out, doses of the univalent penicillin derivative,
the following observations would suggest benzylpenicilloyl-formyl lysine, prior to ad­
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that allergen C, if at all present in KBG-cy­ ministration of penicillin to sensitive pa­

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tochrome c, it would be in trace amounts. tients, was successfully achieved by de

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Cytochrome c of Kentucky Blue Grass Pollen 375

Week and Girard [5J. Alternatively, the al­ allergens. Int. Archs Allergy appl. Immun. 53:
lergenic determinants could be incorporated 162-173 (1977).
9 Ekramoddoullah, A.K.M.; Kisil, F.T.; Sehon,
into tolerogenic conjugates for abrogating
A.H.: Suppression of the IgE antibody re­
the IgE antibody response, as has been sponse in mice to Kentucky blue grass pollen
achieved in experimental animals by the allergens. Int. Archs Allergy appl. Immun. 55:
injection of conjugates of the appropriate 247-254 (1977).
hapten (e.g. benzylpenicilloyl or 2,4-dinitro- 10 Ekramoddoullah, A.K.M.; Chakrabarty, S.;
Kisil, F.T.; Sehon, A.H.; Allergenic cross-reac­
phenyl groups) with nonimmunogenic car­
tivity of two purified allergens isolated from
riers, such as isologous y-globulins [19], po­ Kentucky Blue grass pollen. Proc. Can. Fed.
lyvinyl alcohol [20] or the synthetic copoly­ biol. Soc. 22: 21 (1979).
mer of D-glutamic acid and D-lysine [17]. 11 Ekramoddoullah, A.K.M.; Chakrabarty, S.;
Kisil, F.T.; Sehon, A.H.: Isolation of a purified
allergen from Kentucky blue grass pollen. Int.
Archs Allergy appl. Immun. 63: 220-235
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1 Chakrabarty, S.; Ekrantoddoullah, A.K.M.; A.H.: Isolation of a low molecular weight con­
Kisil, F.T.; Sehon, A.H.: Allergens of Ken­ stituent from Kentucky blue grass pollen pos­
tucky blue grass pollen. II. Isolation of hapten­ sessing carrier activity of high molecular
like components from Kentucky blue grass weight constituent(s). Int. Archs Allergy appl.
pollen by preparative isoelectrofocussing. Int. Immun. 64: 277-286 (1981).
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2 Chakrabarty, S.; Ekrantoddoullah, A.K.M.; Electrophoretic analysis of the major polypep­
Kisil, F.T.; Sehon, A.H.: Isolation and partial tides of human erythrocyte membrane. Bio­
characterization of Allergen C. Int. Archs Al­ chemistry 10: 2606-2616 (1971).
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3 Corradin, G.; Chiller, J.M.: Lymphocyte spe­ J.D.; King, T.P.: Cytochromes c: new ragweed
cificity to protein antigens. II. Fine specificity pollen allergens. Fed. Proc. 38: 1415 (1979).
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rived peptides as antigenic probes. J. exp. Med. with pcrformic acid. J. biol. Chem. 219:
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