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Studies on ergothioneine

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Studies on ergothioneine

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Content STUDIES ON ERGOTHIONEIME
A Thesis
Presented to
the Faculty of the Department of Biochemistry
The University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Master of Science in Biochemistry
k
by
Robert William Ban
January 1956
U N IV E R SITY O F S O U T H E R N C A L IF O R N IA
G R A D U A T E S C HO O L.
U N IV E R S IT Y P A R K
L O S A N G E L E S 7 ^
K5C> B X / X
This thesis, written by
Robert. . WillIammBan
under the guidance of h..~?..Facuity Committee,
and approved by all its members, has been pre
sented to and accepted by the Faculty of the
Graduate School, in partial fulfillment of the
requirements for the degree of
Master of Science
........ S proW
Date..
Harry jP is m . s o.
January 2l*» 195$?“
Faculty Committee
Cmirman
University of Soutnern C altfom ia U&7QITP
TABLE OP CONTENTS j
I
CHAPTER PAGE j
I. HISTORICAL INTRODUCTION 1 |
The Discovery and Structure of ErgothloneIne 1 i
i
Isolation of Ergothlonelne 1 !
Properties of Ergothlonelne 2 i
Determination of Ergothlonelne 3
' i
Occurrence and Distribution of Ergothlonelne 7
Effect of Diet on Blood Ergothlonelne 8 j
i
The Physiology and Biochemistry of Ergo- 9 j
thlonelne j
j
Origin of Ergothlonelne 10
Culture of Clavioeps purpurea 11 j
II. STATEMENT OP THE PROBLEM AND METHOD OP ATTACK 13 !
III. MATERIALS AND METHODS li* '
Blood Analysis lk j
I
Organism 17 1
Culture 18
Extraction of Ergothlonelne 19
Filter Paper Chromat ography 22
Alumina Chromat ography 2$
Growth Studies 28
Histidine Uptake 28
CHAPTER PAGE
IV, EXPERIMENTAL RESULTS AND DISCUSSION 30
Blood Analysis 30
Ergothlonelne Formation in Clavlceps 32
Alumina Chromatography 36
Nutritional Studies 45
Histidine Uptake 51
Growth Studies 54
V. SUMMARY AND CONCLUSION 57
VI. BIBLIOGRAPHY 60
LIST OF TABLES
TABLE
I MEDIA EMPLOYED FOR THE CULTURE OF
CLAVICEPS PURPUREA
II REAGENTS FOR PAPER CHROMATOGRAPHY
III BEHAVIOR OF ERGOTHIONEINE AND RELATED
COMPOUNDS IN FILTER PAPER CHROMATOGRAPHY
IV DETERMINATION OF HISTIDINE
V COMPARISON OF ERGOTHIONEINE BLOOD LEVELS
OF INFANT AND ADULT RATS AND OF THE
ADULT RABBIT
VI COMPARISON OF ERGOTHIONEINE LEVELS OF
ERYTHROCYTE AND RETICULOCYTE FRACTIONS
OF RABBIT BLOOD
VII NINHYDRIN-REACTING AND DIAZ0-REACTING
MATERIAL IN ALUMINA CHROMATOGRAPHY
OF AN EXTRACT OF CLAVICEP3 PURPUREA
VIII ERGOTHIONEINE CONTENT OF CLAVICEPS
PURPUREA
IX GROWTH OF CLAVICEPS PURPUREA IN
DIFFERENT MEDIA
PAGE
20
23
26
29
31
33
1 + 0
1+3
1 + 6
TABLE OP FIGURES
FIGURE
1
2
3
k
5
6
7
8
PAPER CHROMATOGRAPHY OF AN EXTRACT OF
CLAVICEPS PURPUREA
PAPER CHROMATOGRAPHY OF AN EXTRACT OF
CLAVICEPS PURPUREA
ALUMINA CHROMATOGRAPHY OF PURE ERGOTHIO
NEINE AND AN EXTRACT OF CLAVICEPS PURPUREA
SEPARATION OF PURE ERGOTHIONEINE FROM
HISTIDINE BY ALUMINA CHROMATOGRAPHY
ALUMINA CHROMATOGRAPHY OF CLAVICEPS
PURPUREA GROWN ON ACID HYDROLYZED CASEIN
AS THE PRINCIPAL NITROGEN SOURCE
ALUMINA CHROMATOGRAPHY OF CLAVICEPS
PURPUREA GROWN ON A SYNTHETIC AMINO ACID
MIXTURE AS THE PRINCIPAL NITROGEN SOURCE
ALUMINA CHROMATOGRAPHY OF CLAVICEPS
PURPUREA GROWN ON A SYNTHETIC AMINO ACID
MIXTURE WITH HISTIDINE, METHIONINE, AND
CYSTINE ABSENT AND REPLACED WITH AMMONIUM
SULFATE
HISTIDINE DISAPPEARANCE IN A CLAVICEPS
CULTURE
PAGE
35
37
39
1*2
1*8
1*9
50
51
9
GROWTH CURVE OF GLAVlCBPS PURPUREA
55
Chapter I
HISTORICAL INTRODUCTION
The Discovery and Structure of Ergothlonelne
Discrepancies obtained in the determination of
uric acid in blood led workers to suspect the presence
of an interfering substance. Both Benedict (1) and
Hunter and Eagles (2) succeeded in Isolating the com
pound. It was shown by Newton et al. (3) and by Eagles
and Johnson (1+) to be identical with ergothlonelne, a
substance originally isolated from ergot of rye by
Tanret (5, 6, J). Barger and Ewins (8, 9) and Akabari
(10, 11) showed ergothlonelne to be the betaine of
H-N-C-CH2CHCOO- l
HS-c' I I +VN(CH3>3 . The synthe-
N-CH J
sis of ergothlonelne was accomplished in 195>1 by Heath
2-thiolhistidine
et al. (12) who employed histidine as the starting
material.
Isolation of Ergothlonelne
A. Blood
The early methods for the Isolation of ergothio-
neine from blood were complex and time-consuming, and
yields were poor (2). A simplified procedure, with
improved yield, was introduced by Williamson and Mel-
drum (13) who purified ergothioneine as the cuprous
salt. However, Melville et al. (lJ+), using this
method, reported unexpected losses of ergothioneine and
a highly impure final product. Further purification
had to be employed and the final product, although
highly pure, was obtained in very low yield.
B. Ergot
The isolation of ergothioneine from ergot seler-
otia by Tanret (15) was achieved only in low yield.
Its isolation was subsequently improved by Eagles (16)
Pirie (17) and by Hunter et al. (18) who isolated it
in the yield of 0.26$ of the ergot used*
C. Seminal fluid
Mann and Leone (19) purified ergothlonelne as the
phosphotungstate from boar vesicular secretion. The
final isolation depended entirely upon repeated crys
tallization from 70$ ethanol.
Properties of Ergothioneine
Ergothioneine is very unstable in strong alkali.
Boiling with $0$ aqueous potassium hydroxide consti
tutes an effective way of demethylating the compound.
3
Trlmethylamine is evolved and thiolurocanic acid is
produced. Boiling with an aqueous solution of ferric
chloride, dilute nitric aeid, or bromine water results
in the removal of the sulfhydryl group with the pro
duction of histidine betaine and sulfuric acid.
Treatment with excess iodine converts ergothioneine
to the disulfide as an iodine complex. The basic
properties of the imidazole ring of ergothioneine are
modified by the presence of the sulfur atom and there
fore ergothioneine is neutral to litmus.
Determlnation of Ergothioneine
A. Colorimetric estimation
Early methods for the determination of ergothio
neine in blood were based upon the colorimetric reac
tion between ergothioneine and phosphotungstlc acid
(uric acid reagent). The test was applied after the
removal of the uric acid of blood since uric acid
also reacts with this reagent.
Hunter (20) in 1927 introduced a much more
specific test for ergothioneine. According to this
author the test depends upon two main reactions. The
first is the coupling of ergothioneine with the salt
of sulfanilic acid to produce a yellow substance in
k
solution. This substance is very unstable in an ex
cess of weak alkali and the test depends upon the
addition of strong base to produce the second reac
tion, the formation of the highly colored salt, at
a time when the maximum coupling has occurred and
minimum destruction of the coupled product has taken
place. The restriction of the coupling time to thirty
seconds before the addition of base improves the
specificity of the reaction by the elimination of
various potentially interfering substances such as
tyrosine, which yields yellow to orange colors with
the diazo reagent and thereby masks the magenta color
given by ergothioneine, or glutathione, which inhibits
the color formation from ergothioneine itself. Poor
colors are also obtained in the presence of various
inorganic ions, certain reducing substances, alcohol,
or ketones (21). The specificity of the test is gen
eral for the thiolimidazole ring provided it has a free
thiol group in the 2 position and at least one of the
^ or 5 positions unsubstituted or containing a dis
placeable carboxyl group (22). The specificity of
the reaction was further studied by Lawson et al.
(23) who found that of the compounds investigated,
those giving the magenta color were ergothioneine,
2-thiolhistidine, 2-thioluroeanic acid, and ij-phenyl-
2-thiolimidazole.
The preparation of protein-free filtrates from
blood had been an early source of difficulty in the
determination of ergothioneine (Id, 2k, 25). A re
investigation of the problem by Melville and Lubschez
(21) disclosed little or no ergothioneine in blood
filtrates prepared with any of the common protein
precipitants. These workers found that the addition
of glutathione and sodium hydrosulfite to the laked
red cells prior to protein precipitation with tri
chloracetic acid resulted in quantitative recovery
of ergothioneine in the filtrate* This deprotein-
ization procedure was necessary only when dealing
with blood* Other tissues were found to be satis
factorily deproteinized by the use of trichloracetic
acid alone with no loss in ergothioneine*
B* Filter paper chromatography
Paper chromatography has been used for the de
tection of ergothioneine (19* 26, 27* 28). Its
effectiveness in removing interfering substances
which either mask or inhibit the diazo reaction,
! and its sensitivity to microgram quantities make it
particularly useful.
?
| The diazo reagent may be sprayed on the paper
J
!
! separately, followed by a spray of sodium hydroxide
| (27, 28), or the reagents can be mixed and sprayed
|
I on together (26). Ames and Mitchell (28) discuss
t
I the specificity of the diazo reagent on paper towards
1 imidazoles in general. The color produced with ergo-
| thloneine is magenta, with thiolhistidine light orange
i and with histidine and glutathione, yellow. The
j
method will detect approximately a microgram or less
of ergothioneine.
Other sprays which have been used rely on the
, reducing ability of ergothioneine to develop a color
with the reagent employed (26, 29). All these re-
I
! agents lack the specificity of the diazo reagent but
!
I may be used to supplement information on the compound
I in question when used In conjunction with the Rf.
Chromatographic solvent mixtures have included:
butanol, glacial acetic acid, water ($:!p:3)» benzene,
1 butanol, methanol, water (1:1:2:1); propanol, 1 N
acetic acid (3*1), butanol, glacial acetic acid, water
j • • '
t
1 (ii:l:£) and tertiary butanol, water (3:7). The last
two solvent mixtures have been used as the first and
! second solvents, respectively, in a two dimensional
: procedure, (27* 28, 29).
Zinc hydroxide, 80# ethanol, or an acetic acid-
sodium oxalate mixture have been used to prepare
protein-free filtrates prior to chromatography (19* 29)
i For best results it was found necessary to remove salts
by means of a cationic resin before chromatography (29)
G. Column chromatography
The removal of interfering substances before
applying the diazo test was attempted by Melville and
Horner (30), who chromatographed protein-free blood
| filtrates on alumina. Modifications were later made
I
by Melville et al. (31) to improve yields, increase
the sharpness of the elution bands, and decrease the
time required for carrying out the analyses. Khen
! applied to blood the method appears to give quantita-
I tive results.
i
I . . . .
i Occurrence and Distribution of Ergothioneine
' The known occurrence of ergothioneine remained
limited to the ergot fungus and blood until recently,
when it was discovered in boar seminal vesicle secre
tory fluid and was shown to be widely distributed in
8
mammalian tissues, being particularly rich in liver
and kidney (19* 31).
Although it is known that the ingestion of cer
tain grains can cause marked increases in blood ergo
thioneine levels, ergothioneine has never been found
in higher plants.
The presence of ergothioneine in fungi other
than Glavlceps purpurea was established by Melville
in 1955 (32). He found ergothioneine in Aspergillus
and Altonaria.
Effect of Diet on Blood Ergothioneine
The pronounced effect of diet on the blood ergo-
thioneine levels of animals early became apparent.
Animals maintained on purified diets had no detectable
blood ergothioneine, but when these diets were sup
plemented with certain grains or with liver extract,
relatively high levels were found (2$, 33* 3^)«
Although vitamin B^2 can increase the blood level
of non-protein sulfhydryl groups this vitamin has no
effect on blood ergothioneine levels (35* 36).
It appears from the recent work of Melville et al.
(l4) that the presence of a precursor in the diet is
unlikely and that ergothioneine may be present in the
9
diet in extremely low concentration but is neverthe
less accumulated by the animal.
The Physiology and Biochemistry of Irgothloneine
Most of the findings on physiological effects
have been negative, equivocal or controversial, and
are difficult to fit into any theory of the biochem
ical role of ergothioneine. Apparently ergothioneine
is rather inactive pharmacologically (37» 3©). It
was claimed by Lawson and Rlmington (39) that ergo
thioneine exerted an antithyroid effect but this
work could not be confirmed and was later retracted
by the same authors (1+0). Significant Increases in
ergothlonelne blood levels have been claimed to occur
in diabetic patients (ill). However the production of
alloxan diabetes was found by Beatty (1^2) to have no
affect on blood ergothioneine levels in the rat.
Clinical studies have shown significant variation in
mean values in patients of different ages and with
various pathological conditions (ij.3* Mj-K A possible
role of blood ergothioneine wa3 indicated by the work
of Spicer et al. (3^) who showed that ergothioneine
could protect against nitrite-induced methemoglobinemia.
The presence of ergothioneine in boar seminal fluid
10
I apparently serves to maintain the reducing properties
j of boar seminal ejaculate, thereby serving to protect
; the sperm from oxidation (19).
Heath et al. (26) found that ergothioneine is
; rapidly accumulated in the liver and more slowly In
the erythrocytes of rats receiving ergothioneine in
the diet. Ergothioneine was also found by these
workers to be partly rapidly excreted in the urine of
i
! rats receiving ergothioneine either orally or subcu-
i ' ■ ......... • ■ ■ ' !
| taneously. No ergothioneine was detected in the plasma, i
i j
| The presence of ergothioneine normally in urine j
j j
was claimed by Work (i+J>). This was later disproven by 1
the work of Lawson at al. (1+6).
Origin of Ergothioneine
A. Radioisotope studies
Attempts to find whether ergothioneine can be
synthesized in the animal have given conflicting
results. Heath et al. (1+7) and Heath (1+8) found that
S^-methionlne could label ergothioneine but that
; neither S^-sulfate nor S^-thiolhistidine acts as
a source of ergothioneine sulfur in the boar.
Melville et al. (ll+, 1+9)» however, were unable
to find labeled ergothioneine when S^-methionine
11
was administered to the pig, rat and guinea pig or
| when S^-cystlne was administered to humans. Carbon
precursors such as 2-C^-glycine, 2-C^-histidine,
C^-methyl methionine and C^-formaldehyde were
studied, but these workers could find no labeling
in the ergothioneine in the rat, chicken, guinea pig
or human. It was the opinion of this group that
animal ergothioneine is of dietary origin.
Culture of Claviceps purpurea
' The ergot fungus Claviceps purpurea grows as a
I
parasite on rye and other grasses, producing sclerotia
which are the only known source of ergot alkaloids.
' As the medical importance of these alkaloids increased,
the possibility of culturing the fungus under controlled
conditions in the laboratory prompted numerous workers
to investigate nutritional and environmental factors
*
' in relation to their effect on growth and alkaloid
I
| production in this organism. No attention was given
to the possible presence of ergothioneine under these
conditions.
i
Early investigations of this organism were limited
by the slow growth whieh the organism exhibited on
surface cultures. Tyler and Schwarting (50) succeeded
12
in developing a nutrient medium in which the organism
could produce a near-maximum amount of growth in a
relatively short period of time. Maximum growth,
using submerged culture in 500 ml. Erlenmeyer flasks
shaken on a reciprocating shaking machine, was reached
in a period of approximately seven days. The culture
medium consisted of a salt solution, mannitol as the
sugar source, and a casein hydrolysate as the principal
nitrogen source.
Chapter II
STATEMENT OP THE PROBLEM AND METHOD OP ATTACK
Despite a renewal of interest in the problem of
the origin and role of ergothioneine at the time this
project was undertaken, little progress had been made
j in establishing whether a direct precursor or ergothio-
J nelne Itself is ingested in the diet. It had not been
| established whether animals could synthesise this com-
i
pound de novo and its function was and still is quite
j obscure*
1 The ultimate goal of the study to be reported was
1 ' . . . . . . .
; to gain an understanding of the origin of ergothlonelne.
i . . . . . .
1
| A biological system was sought, therefore, in which ergo-
, thionelne could be demonstrated to occur and which could
be conveniently studied in order to determine how this
j takes place*
J As stated previously, ergothioneine was originally
discovered in the ergot fungus, Claviceps purpurea,and
j
ha3 been shown to be present in relatively high concen-
j . . . . . . . . . '
i tration in ergot sclerotia. This organism is, therefore,
| a logical choice to consider in studying ergothioneine
i biosynthesis.
Chapter III
MATERIALS AND METHODS
Blood Analysis
A. Rat
Blood, obtained from adult and eig^it-day-old rats
by heart puncture, was analyzed by the procedure of
Melville and Lubschez (21) as described:
Reagents:
Trichloracetic acid, reagent grade, used as a 35$
solution in water, freshly prepared.
Chloroform, reagent grade, washed 3ix times with equal
volumes of distilled water, stored in dark glass bottle
Sodium hydrosulfite, powdered (Malllnckrodt).
Glutathione. (Schwarz Laboratories, Inc., New York),
aqueous solution, 35mg« P©** ml*, freshly prepared.
Ion exchange resin, Amberlite IRA-UlO (The Resinous
Products Division, Rohm and Haas Company, Philadelphia)
Prepared by shaking 500 gm. of the commercial moist
chloride salt with 2 liters of 5$ NagGO^ for 3 hours,
washing with distilled water until free from alkali,
and collecting on a funnel with the aid of suction.
Alkaline buffer solution, sodium carbonate, reagent
grade, anhydrous (200 gm.) and sodium citrate dihydrate
' (57 ) were dissolved in approximately 800 ml, of
| water with heating. The solution was cooled, filtered
by gravity, and diluted to 1 liter* The solution was
stored in polyethylene containers.
Sulfanillc acid, reagent grade, 0,2$ solution in 1$ HC1.
! Sodium nitrite, reagent grade, solution in water.
Diaaotlzed sulfanllic acid, one volume of cold NaN02
solution, mixed with 10 volumes of cold sulfanilic
acid solution, and allowed to stand 10 minutes before
| . • ■
i using, kept in an ice bath, freshly prepared,
19 N Sodium hydroxide, prepared from reagent grade NaOH
; filtered through a sintered glass funnel, stored in
polyethylene containers.
Procedure:
1 One ml, of heparlnized blood was delivered into
I a 12 ml. graduated centrifuge tube containing 10 ml. of
J 0.9$ NaCl solution. The blood was thoroughly mixed
with the salt solution and the mixture was centrifuged,
| As much as possible of the diluted plasma was removed,
; Water was added to the 1.0 ml, mark and the mixture was
stirred with a glass rod to facilitate laking. Then 0.£>
; ml. of a freshly prepared solution of glutathione con-
16
tainlng 35 mg. per ml. was added and the solutions
were well mixed. Approximately 25 mg. of solid sodium
hydrosulfite were added and the mixture stirred. The
tube contents were mixed with 5*0 ml. of water and the
proteins were precipitated by the addition of 0.5 ml.
of 35$ trichloracetic acid. The well stirred mixture
was centrifuged, and the supernatant solution was poured
into a small separatory funnel and shaken with 15 ml.
of washed chloroform. The chloroform phase and any
solid material were discarded, and the aqueous layer
was transferred to a glass-stoppered tube of 15 ml.
capacity. Approximately ! { . ml. (about 2.1* gm.) of moist
ion exchange resin were added. The tube was stoppered
and shaken mechanically for 10 minutes. The solution
was separated from the resin by gravity filtration
through a funnel containing a small plug of absorbent
cotton. A 3 ml. aliquot of the ehloroform-and resin-
treated filtrate was transferred to a test tube. The
tube was placed in an ice bath, 0.5 ml. of alkaline
buffer solution was added, and the solutions were
mixed. To the cold solution 0.5 ml. of dlazotlzed
sulfanilic acid was added, and the solutions were
mixed and allowed to stand in the ice bath for J +5
seconds. Then 1.0 ml. of 19 N NaOH was added rapidly,
17
and the solutions were mixed and allowed to stand until
free of gas bubbles. The samples were read in the
Coleman Junior spectrophotometer at $\\Q m •
B* Rabbit
Blood, obtained by heart puncture, was analyzed
as mentioned above. The cells from 2$ ml. of blood
were separated from the plasma by centrifugation. The
cells were separated into reticulocyte and erythrocyte
fractions. The denser reticulocytes pack at the bottom
of the cell mass, while the upper layer contains the
majority of the erythrocytes. Accordingly, the upper
half was separated, resuspended in saline and recentri
fuged. The upper half of the resulting sediment was
separated resuspended in saline and again recentrifuged.
The upper half of the resulting sediment was separated
and saved for analysis. An analogous differential cen
trifugation was used to obtain a retieulocyte-enriched
fraction from the bottom layer of the original cell
mass. An equal volume of cells was taken from each
tube for analysis.
Organism
Claviceps purpurea No. 9605, C. L. Lefebre, U. S.
D. A., was obtained from the American Type Culture
18
Collection, 2029 M Street, N. W., Washington, D. C.
Upon receipt of the culture the organism was subcultured
on Sabouraud agar slants* These subcultures were then
examined microscopically. They appeared to be free
from contamination. Pieces of surface mycelium were
then sterilely transferred to liquid culture medium.
Culture
The culture medium developed by Tyler and Schwar-
tlng (51) was used initially. The composition of this
medium is shown in Table I. Protolysate, manufactured
by Mead Johnson & Co., Evansville, Indiana, was used
to supply the casein hydrolysate.
The fungi were adapted to the medium by serial
transfers. Pour transfers were sufficient. Five
hundred-ml. Erlenmeyer flasks containing 1+0 ml. of
medium were used routinely. Since faster growth
occurred when the flasks were continuously agitated,
the organism was cultured in flasks which were shaken
at the rate of 80 cycles per minute on a shaking
machine.
Claviceps was also cultured on Czapek's medium,
which has an inorganic source of nitrogen only, and
on modifications of Tyler and Schwarting* s medium
19
wherein the casein hydrolysate was replaced as follows:
(1) a synthetic amino acid mixture simulating the casein
hydrolysate, (2) the same amino acid mixture with histi
dine and with histidine, methionine, and cystine omitted j
and replaced by an equivalent amount of ammonium sulfate,
(3) & simplified mixture of amino acids found by Tyler
and Schwarting (51) to produce satisfactory growth, and
(Ij.) asparagine supplying the principal nitrogen source.
The composition of these media is shown in Table I.
Extraction of Ergothioneine
The fungal mycelium was filtered from the medium
on a Buchner funnel and washed with distilled water.
The material was then placed in a vacuum desiccator
until completely dry. Ten grams of dried fungus were
cut up with scissors and refluxed for three hours in
I 4 .O ml. of 80$ ethanol. The material was filtered
through Whatman No. 50 filter paper on a Buchner funnel
while still hot. This extract was then concentrated
under vacuum to a final volume of 2 ml. The precipitate
which formed was centrifuged down and the supernatant
was taken for analysis. The use of hot water alone for
extraction resulted in the presence of too much protein
and other material in the extract. When triehloracetic
Table I
20
MEDIA EMPLOYED FOR THE CULTURE OF CLAVICEPS PURPUREA
A. Medium of Tyler and Schwarting
19Hi.N0.
U 3
kh2pou
1.0 gnu
2.0 gm.
.7H20 0*5 gm.
CaCl2 0.1 gm.
FeSOjj. 7HgO 0.1 gm.
Na2Bi|°7*10H20
CuS0^.5H20
MnClo.i|Ho0
0.088 mg.
0,392 mg.
0.072 mg.
{m^)6uo7o2^ k ^ 2° °*°37 mS«
ZnS0j +*7H20 8.8l mg,
Mannitol, 20.0 gm.; casein hydrolysate, 10.0 gm.;
distilled water, to make 1 liter.
Casein hydrolysate of above medium replaced with
the following amino acids:
glycine 0.05 gm. L-glutamic acid 3.0 gm.
DL-alanine 0.20 gm. L-aspartic acid
0
-3*
.
0
gm.
DL-valine 0.80 gm. L-proline 0.90 gm.
DL-leucine 0.50 gm. DL-serine
0
UN
.
O
gm.
L-isoleucine 0.50 gm. L-cystine
0.05
gm.
DL-phe ny1alanine 0.i40 gm. DL-methionine 0.60 gm.
L-tyrosine 0.50 gm. L-histidine.HCl
O
.
O
gm.
L-tryptophane 0.20 gm. L-lysine.HCl
CM
t-
.
O
gm.
L-threonine O.I4O gm. DL-arginine.HC1
00
.
O
gm.
Table I (continued)
21
C. Two modifications of the amino acid mixture were
used: (a) histidine was omitted (b) histidine,
methionine, and cystine were omitted and replaced
by 0.60 gm. of (NHjjJgSO^ per liter.
D. The amino acid mixture was simplified:
DL-alanine 1.0 gm. L-glutamic acid 1,0 gm.
L-asparagine 1.0 gm. L-leucine 1.0 gm.
L-aspartic acid 1.0 gm. DL-valine 1.0 gm.
E. Ten grams of asparagine per liter were used to
replace the amino acids used in the above mixture.
P. Czapek's medium
sucrose 30.0 gm. MgS0|| 0.5 gm»
NaNO-j 3.0 gm, KCl 0.5 gm.
K2HP0Jj 1.0 gm. FeSOjj 10.0 rag.
Distilled water to make 1 liter.
22
|
j acid was used to deproteinize, no ergothioneine could
i be^ detected on the chromatograms, perhaps because of
I
! the excessive concentration taking place after the
filtrate was reduced in volume for application on paper.
!
Filter paper chromatography
j Ascending paper chromatography was used to separate
j the compounds of the fungal extract. The solvent system
! used initially was butanol, glacial acetic acid, water
! (14:1:5). This was prepared fresh when used. The chroma-!
|
I tograms were run on Whatman paper No. I 4IH. This paper ;
| I
is strongly resistant to the action of alkali. j
•t
; Color development of the spots was obtained initially
j . . . . .
by spraying with a solution of diazotized sulfanilic
acid followed by a spray of sodium hydroxide solution.
Later the spray used consisted of a mixture of equal
j parts of diazotized sulfanilic acid and 20# sodium
j hydroxide solution. The diazo reagent was also modified
; for use as a dipping reagent. The composition and
! preparation of these reagents is given in Table II.
, The paper must be completely free of solvent before
| the spray is applied. If not, a background color will
I
develop which may become quite intense, masking the
. . . . . .
i
1 spots obtained. Whatman paper No. 1j.1H appears to be
23
Table II .
REAGENTS FOR PAPER CHROMATOGRAPHY
; Reagents: Diazo Spray ReaSent
Sulfarilllc acid solution, 0.9$ in a solution containing
9 ml. concentrated HC1 per 100 ml.
! Sodium nitrite solution, 5$ in water.
I ' v .
I Sodium carbonate, anhydrous, 1 gm.
! Sodium acetate, anhydrous, 10 gm.
I . . . . . .
| Sodium hydroxide, 10 N solution
i
| Preparation:
Diazotized sulfanilic acid. In a $0 ml. volumetric flask
i
i immersed in ice 1.$ ml. of the sulfanilic acid solution
was added, followed by 1.5 ml. of the sodium nitrite solu-
| tion. The mixture was allowed to stand for 5 minutes,
i . . . . . . . : ■ " ’
then a further 6 ml. of the nitrite solution was added.
After an additional 5 minutes ice cold water was added
I to the mark and the contents were mixed.
| Alkaline buffer. One gm. of anhydrous sodium carbonate
I • ■
and 10 gm. of anhydrous sodium acetate were dissolved
in water and the volume made up to 100 ml.
Piaz° reagent. Equal parts of the buffer and the diazo-
! tized sulfanilic acid were mixed and used as a spray,
* .
i 10 N Sodium hydroxide. Sprayed on after the diazo spray.
2k
Table II (continued) ;
j
Diazo Spray Reagent (19)
Reagents;
Sulfanilic acid solution, 0.9# in a solution containing
9 ml, concentrated HC1 per 100 ml, !
Sodium nitrite solution, 5# In water.
Sodium hydroxide solution, 20# in water.
i
Preparation;
Diazo reagent. Two parts of the sulfanilic acid solution j
i
were mixed with one part of the sodium nitrite solution j
i
- !
at 0 ; allowed to stand in the cold for 10 minutes. \
Spray reagent. One part of the diazo reagent was mixed j
with one part of the 20# sodium hydroxide solution and
the solution was immediately sprayed on the paper.
Dipping modification. A solution of 20# sodium hydroxide
in 15# ethanol was substituted for the 20# sodium hydrox- >
ide solution used in the preparation of the spray reagent.
Folin-Marenzi Reagent (52)
Reagents: j
Sodium hydroxide solution, 10# in water.
Phosphotungstlc Acid (Folin-Marenzi Reagent), prepared
as described by Folin and Marenzi (53 )•
Procedure:
The paper was first sprayed with 10# sodium hydroxide i
solution and then with the phosphotungstlc acid solution. ;
25
the best suited for use with this reagent, since not
only is it resistant to the action of alkali but also
it does not develop background coloration as do other
papers Investigated, e*g» Whatman No. 1.
The Folin-Marenzi reagent, shown in Table II, as
employed by Work (52), was also used as a spray to
supplement Information on the nature of the spots ob
tained* The color and Rf values of the compounds con
sidered using the diazo and Folin-Marenzi reagents and
the solvent; system previously described are shown in
Table III.
After extraction and concentration as mentioned,
an application of 0.01 ml. of extract per spot was
sufficient for good color development of the spots.
As little as 1 microgram of ergothioneine could be
detected by this procedure.
Later the solvent system, propanol, 1 N acetic
acid (3:1) was adopted and used routinely since it
gave somewhat better resolution of these substances.
The Rf values obtained with this solvent system are
also listed In Table III.
Alumina chromatography
Alumina columns were used to fractionate fungal
Table III
BEHAVIOR OP ERGOTHIONEINE AND RELATED COMPOUNDS IN FILTER PAPER CHROMATOGRAPHY
Compound Rf wltli (3:1) R^ with Xl|Vl:5) Color developed In:
propanol-1 N acetic butanol-acetic Diazo Polln-
acid acid-water test Marenzi
test
Histidine 0.12 0.13 yellow no reaction
2-Thiolhistidine 0.15 0.15
orange blue
Ergothioneine 0. 27 0.25
magenta blue
2?
extracts since more accurate determination of larger
quantities of ergothioneine was desired. The method
used was substantially that of Melville et al. (31)•
Alcoa F-20 alumina was initially employed. The alumina
was first washed with distilled water and the superna
tant decanted until no more turbidity was noted in the
wash water. The alumina was then dried in the oven at
, . .. £
200° overnight. Twenty grams were moistened with
column solvent consisting of 75$ aqueous ethanol con
taining 1 ml. of formic acid per 100 ml. and packed as
a slurry in a column of 9 mm. inside diameter. The
ethanol used was prepared by distilling it off of
sodium metal. Harshaw Scientific activated, powdered
catalyst grade A1-0101P alumina was found to give better
resolution than the Alcoa grade and was subsequently
used in its place. It was not found necessary to wash
the Harshaw grade alumina before use.
The sample was placed on the column in 5 ml. of
75$ aqueous ethanol. From 3 to 5 ml* fractions were
collected using gravity flow at a rate of ml. per
hour on a Misco time flow counter. One ml. aliquots
were removed and evaporated to dryness with a stream
of hot air. Three ml. of water were added, followed
by 0.5 ml. of a carbonate-citrate buffer and 0.5 ml. of
28
diazo reagent. One ml. of 19 N sodium hydroxide was
added i *5 seconds after the addition of the diazo re
agent* All solutions were kept chilled in an ice bath.
When the solution had cleared of bubbles, the color was
read in the Klett using a No. $k g**een filter.
Growth studies
Growth of the cultures was measured by recording
the dry weight produced at different time intervals.
Growth was measured in 3>0- and 250 ml. Erlenmeyer flasks
containing 5- and 10 ml. of medium, respectively.
Histidine uptake
Ten grams of wet fungus were washed twice in sterile
20$ mannitol solution. The fungus was then sterilely
transferred to a 250 ml. Erlenmeyer flask containing 10
ml. of a solution of 20 mg. of mannitol and 2.5 mg. of
histidine per ml. At hourly intervals an aliquot of
0.01 ml. was removed and analyzed by the Macphbrson
modification of the Pauly method for the determination
of histidine (54)5* The color produced was read in the
Klett-Summerson colorimeter with a No. 5U green filter.
The procedure is shown in Table IV.
Table IV
DETERMINATION OP HISTIDINE (5i|)
Reagents:
Sulfanilic acid solution, 1# in 10# HC1.
Sodium nitrite solution, 5# in water*
Sodium carbonate solution, 30# in water.
Absolute ethanol, 10 ml.
Procedure:
1, Add sample in 1.0 ml, of water,
2, Add 1 ml* of sulfanilic acid solution.
3, Add 1 ml. of sodium nitrite solution.
i*. Mix and let stand at room temperature for 30 min
5. Add 3 ml. 3odium carbonate solution, and mix.
6. Add 10 ml. ethanol.
7. Cool under tap.
8. Dilute to 30 with water.
9. Read in Klett with No. SkO filter.
| Chapter IV
I
I
j EXPERIMENTAL RESULTS AND DISCUSSION
i
t
Blood Analysis
. 1 — l i [ -.nirnir-iiMi-
A* Rat
The blood ergothioneine levels of adult and infant
rats were compared. The blood level of the adult rat
| was found to be 6 mg. per 100 ml. while no ergothio-
t
! neine could be detected in the blood of eight-day-old
i
rats. The results are shown in Table V.
Since the rat shows striking increases in ergo
thioneine levels during growth it should be suitable
for studies on the origin of ergothioneine. It would
appear likely that the increased ergothioneine found
! after maturation of the rat is due to the change in
i • • • - • -
| diet rather than to a different metabolic state (see
! page 8)* These results substantiate those found re-
| cently by Beatty (1+2), who observed that the level of
i
t
blood ergothioneine In the rat increases with age and
l
S reaches a plateau at about 6 months.
i
i R a b b i t
I The blood ergothioneine level was determined on
■ a single rabbit (Table V) and was found to be approx-
Table V
31
COMPARISON OF ERGOTHIONEINE
BLOOD LEVELS OF INFANT AND
ADULT RATS AND OF THE ADULT
RABBIT
Sample Quantity Absorbance
Ergothioneine 10/«gm.
0.185
20 " 0.21+0
30 " 0.350
1*0 M 0.1+60
80 « 0.920
Adult Rat 1.0 ml. 0.1+70
Infant Rat
(8-day-old)
1.0 ml.
0.005
Adult Rabbit 1.0 ml. 0.310
Concentration
6,0 mg./lOO ml.
{ 0.03 mg./lOO ml,
J 4 ..O mg./lOO ml.
Analyses were performed by the Melville et al, (21)
modification of the Hunter technique as previously
described. Blood analyses were carried out on the
cells separated from the indicated volume of whole
blood. Absorbances were read in the Coleman Junior
spectrophotometer at a wavelength of 51+0 mp.
32
imately 1 | mg. per 100 ml., or somewhat lower than in the
rat. Since the previous nutritional history of the
rabbit used was not known, it is possible that the dif
ference in levels was not a true species difference but
was due to the diet.
The apparantly complete absence of ergothioneine
from blood plasma suggested that it might be incorpor
ated into the red blood cell at an early stage of ery-
thropoiesis* To test this possibility, a sample of
rabbit blood was fractionated into predominantly re
ticulocyte and erythrocyte portions, respectively*
As shown in Table VI, no significant difference in
ergothioneine levels could be detected between these
fractions. The values obtained indicate the presence
of ergothioneine in the early stages of hematopoiesis.
Since ergothioneine is present before the maturation
of the retleulocyte, a noticeable affect on erythro
cyte levels would not be expected when administering
suspected dietary precursors until newly synthesized
cells have appeared in the blood in significant
numbers.
Ergothioneine Formation in Glavlceps
Dried mycelium from fungi grown on the Protolysate-
33
Table VI
i
comparison op ergothioneine levels op i
ERYTHROCYTE AND RETICULOCYTE FRACTIONS !
OF RABBIT BLOOD j
i
i
Sample Quantity Absorbance Concentration j
Reticulocyte fraction 1.0 ml. 0.310 i+.O mg./lOO ml. 1
i
Erythrocyte fraction 1.0 ml. 0.310 1+.0 mg./lOO ml.
i
Analyses were performed by the Melville et al. (21)
modification of the Hunter technique as previously
described. Twenty-five ml. of rabbit blood was frac
tionated into erythrocyte and reticulocyte enriched
portions as previously described. The cells consti
tuted 1+0$ of the volume of the blood sample. Therefore j
0.1+0 ml. of packed cells, representing the amount present !
1
in 1 ml. of whole blood, was taken for analysis. Absorb- j
ances were read in the Coleman Junior spectrophotometer
at a wavelength of $1+0 mji*
I
i
1
i
1
!
/
3k
mannitol medium was extracted as previously described*
Aliquots of 0.01 to 0.10 ml. of extract were chromato-
i
I ■ '
! graphed together with authentic standards containing
i
| 10 micrograms of ergothioneine. The solvent system
!
j used was butanol, glacial acetic acid, water (ij.:l:5).
1 ' '
j After spraying the strip with the diazo reagent, three
j yellow spots were observed which had originated from
| the extract. Only one of these spots changed to the
characteristic magenta color of ergothioneine when the
strip was next sprayed with alkali. This spot appeared
at Rf 0.25 both for the standard ergothioneine spot and
for the extract. The other two spots which were yellow
were identified as histidine at Rf 0.13 and glutathione
at 0.33. In a second experiment, portions of the
extract with and without added ergothioneine were
chromatographed together with a standard spot of ergo
thioneine. The spots obtained from co-chromatographing
the extract confirmed the previous results (see Figure 11
Further support for the identification of the spot
at Rf 0.25 as ergothioneine was obtained by spraying
similar strips with the Folin-Marenzi reagent. Strips
sprayed with this reagent showed only two blue spots.
One of these spots, at R^ 0.25, corresponded to ergo-
i
thioneine. The other spot corresponded to glutathione
35
Figure 1
PAPER CHROMATOGRAPHY OF AN EXTRACT
OF CLAVICEPS PURPUREA
Diagrammatic:
solvent
front
0
0 glutathione
0
O 0 ergothioneine
0
©
histidine
" S J L \J
origin
• • I ' •
extract extract ergothio-
with neine
added
ergo
thioneine
Solvent: butanol, glacial acetic acid, water (5:1:1+)
Color reagent: diazotized sulfanilic acid, 20$ NaOH (1:1)
36
at Rf 0.33* This spot had also been found on the
chromatograms sprayed with the diazo reagent. Figure
2 shows these results.
The possibility that ergothioneine was not being
biosynthesized at all, but was present in the admin
istered medium was tested by chromatographing a series
of solutions of Protolysate alone. No ergothioneine
was detected using filter paper chromatography as
described above. A series of solutions of culture
medium after incubation with fungus were also paper
chromatographed but no ergothioneine was detected with
the diazo test.
Despite the sizeable quantities of ergothioneine
within the mycelia its absence from the medium suggests
that it can either be actively concentrated in the cells
or that it cannot permeate the cell membrane. In ani
mals ergothioneine is similarly found in the red cells
of the blood but not in the plasma. Since hydrolysis
is not required for extracting ergothioneine from dried
mycelia it can therefore be considered to be present in
the free form.
Alumina chromatography
The behavior of pure ergothioneine and an extract
37
Figure 2
PAPER CHROMATOGRAPHY OP AN EXTRACT
OP CLAVICEPS PURPUREA
Dia grammat i g
extract extract with ergo-
added ergo- thioneine
thioneine
solvent
front
glutathione
ergothioneine
origin
Solvent: butanol, glacial acetic acid, water (^;1sI4)
Color reagent: 20# NaOH followed by the Polin-Marenzi
reagent
38
°** Clavicepa purpurea on alumina columns, prepared and
used as described in Chapter III is compared in Figure 3*
I
Ergothioneine was eluted from the columns in a relative
ly wide range of 20 to 1*5 ml* of effluent, with a peak
I
occurring between 2$ and 35 *nl«
The limiting load of the columns was approximately
1 mg. of ergothioneine. Larger amounts caused tailing
to occur*
Vllhile the characteristic magenta color for ergo
thioneine appeared for pure ergothioneine chromatograph
ed on alumina, orange colors usually were observed when
i
fungal extracts were chromatographed*
I
Upon addition of alkali in the diazo test, the
odor of trimethylamine was evolved, a property charac
teristic of ergothioneine. The odor corresponded to
the fractions which were found to give orange colors.
Vllhile Melville et al. (31) claim that chromatography
on alumina frees ergothioneine from all ninhydrin-
reacting material, definite contamination with such
material was observed in our fractionations. This is
shown in Table VII. The ethanolic fungal extract was
not purified prior to applying it to the column* This
prior purification forms part of the Melville procedure
for blood, but is claimed to be unnecessary for extracts
39
Figure 3
ALUMINA CHROMATOGRAPHY OF PURE ERGOTHIONEINE
AND AN EXTRACT OF CLAVICEPS PURPUREA
200
180
160
li+O
Klett
120
units
100
80
60
1 + 0
20
Pure ergothioneine: 300 micrograms (0.3 ml. of 1.0$
solution of ergothioneine in 75$ ethanol), 1 ml. ali
quots of 5 ml. effluent fractions were taken for ana
lysis, flow rate was 1+ ml. per hour
Extract: a 75$ ethanolic extract of 0.28 gm. of dried
fungus in 10 ml., 5 *fll« of extract was placed on the
column, 2 ml. aliquots of each 5 ml. effluent fraction
was taken for analysis, flow rate was 1+ ml. per hour
pure
ergo
thioneine
extract
ml. of column effluent
5o
Table VII
NINBYDRIN-REACTING AND DIAZO-REAGTING
MATERIAL IN ALUMINA CHROMATOGRAPHS
OP AN EXTRACT OP GLAVICEPS PURPUREA
Ml. of Column Diazo Ninhydrin
Effluent Reaction Reaction
5 * *
10
15 - 4
20 4 4
25 4 t
30 4
35 4 4
ho 4 t
I45 . 4
50 - 4
55 * 4
60 * 4
65 - 4
- 7© - 4
i+ 1
of other animal tissues, and was therefore omitted in
our procedure. Such a purification step might have
resulted in improved resolution.
It was suspected that histidine was responsible
in part for the orange coloration by contributing a
yellow component in the diazo test. That this was the
case for the last of the ergothioneine-containing efflu
ent fractions is shown in Figure i+. No attempt was
made to improve the separation of the yellow component
from the ergothioneine, although if smaller fractions
were taken (1 ml.) it was possible to obtain an aliquot
which gave a magenta color. Instead paper chromatography
was used to confirm the presence of.ergothioneine in
the 20-1+5 range. A portion of the ergothioneine
peak was concentrated and chromatographed on paper using
propanol, 1 N acetic acid (3*1) as the solvent system.
Co-chromatography verified the presence of ergothioneine.
Having identified ergothioneine in the 20 to 1+5 i»l*
effluent range, an estimate of the ergothioneine content
Claviceps was made on the basis of the Hunter reaction
read in a Coleman Junior spectrophotometer at 51+0
The content of ergothioneine was estimated to be 100 mg.
per 100 gm. of dry fungus as shown in Table ¥111.
Because of the obvious contamination of the ergo-
Figure I 4
SEPARATION OF PURE ERGOTHIONEINE FROM
HISTIDINE BY ALUMINA CHROMATOGRAPHY
Klett
units
100
90
80
70
e rgothione ine
Histidine
10
ml. of column effluent
Pure ergothioneine: 100 micrograms (0.1 ml. of l.i
solution of ergothioneine in 75$ ethanol);
Pure histidine: 100 mierograms (0.1 ml. of 1.0$ solution
of histidine In 75$ ethanol);
The samples were mixed and placed on the same column in
5 ml. of 75$ ethanol. One ml. aliquots of each 3 ml.
effluent fraction were taken for analysis. The flow
rate was 1* ml. per hour.
1 + 3
Table VIII
ERGOTHIONEINE CONTENT OF CLAVICEPS PURPUREA1
Fraction Klett Units Standard
(micro-
grams)
Klett Units
H
3 5 1+5
12
1+9
10 85
13
7 k
20 172
ill 121 1+0
355
7*
16
Average;
1+7
9*0 Klett Units per
micrograra
18 5
_______ 3. li5Q x 2 z 100 micro-
3 9 grams from
1+50 0.1 gm. of
dry fungus
1. Mycelium was harvested by the usual procedure,
dried and extracted with 75$ ethanol. The ex
tract from 0.10 gram of dried mycelium was evap'
orated to dryness and taken up in 5 of 75$
ethanol. This entire solution was placed on
the column and eluted with 75% aqueous ethanol
containing 1.0$ formic acid.
2. 1 ml. aliquots of 2 ml. effluent fractions were
taken for analysis.
thibneine peak with one or more nlnhydrin-reactlng com-
I
| pounds, at least one of which produces a yellow color
I In the Hunter reaction, the procedure described above
i cannot be relied upon for the Isolation of radiopure
! ergothionelne in an extract of fungus incubated with
i 1 ) 1
labeled substrates* The evidence presented in Pig-
ure ii that histidine contaminates at least the tail of
{ . . T ...
!
1 the ergothioneine peak would make the procedure partic-
! ularly unsuited for experiments where labeled histidine
is administered* On the basis of the results shown
i in Figure I j . it would appear that histidine contamination
could be eliminated by rejecting the 3$-k$ ml* portion
! of the ergothioneine range. However adsorption chroma
tography separations are notoriously susceptible to the
presence of extraneous adsorbable Impurities, and it
must be considered probable that the separation of his
tidine from ergothioneine from a fungal extract is even
; t
less complete than it is from a synthetic mixture of the
two. If alumina chromatography is to be used at all
for such experiments, it will clearly be necessary to
introduce an additional purification step either pre-
i
;
ceding or following the adsorption on alumina. Since
the choice of alumina was based on its reported ability
to separate ergothioneine from ninhydrin-reacting mater-
h $
ials, an entirely different purification scheme may
prove to be more satisfactory.
Nutritional Studies
Claviceps was cultured on different media in order
to determine whether ergothioneine biosynthesis could
be influenced in this way.
Although growth occurred on Gzapek*s medium, in
which the nitrogen content is solely in the form of
nitrate, the growth was extremely slow. This was also
the case when asparagine was used to supply the only
organic source of nitrogen. Growth on these nitrogen
sources was so slow as to exclude any further work
with the fungus grown on the above media.
Growth was measured as the amount of dry weight
obtained at 100 hours of incubation. Equal and maxi
mum growth occurred on media in which the main source
of nitrogen was supplied either by: Protolysate (an
enzymic digest of casein), an acid casein hydrolysate,
a synthetic mixture of amino acids paralleling the
amino acid content of casein, and the same mixture with
histidine and with histidine, methionine, and cystine
withdrawn and replaced by an equivalent amount of
ammonium sulfate (see TableIX }.
1 * 6
Table IX
GROWTH OP CLAVICEPS PURPUREA
ON DIFFERENT MEDIA
Nitrogen Source Growth Rate
(Mg./lOO Hr.)
potassium nitrate extremely slow
asparagine 1
six amino acids
(see Table I, D., pg. 20)
Protolysate
(enzymic casein digest)
acid hydrolyzed casein
synthetic amino acid mixture
(see Table I, B., pg. 20)
same anilho acid mixture with
histidine withdrawn
same amino acid mixture with
histidine, methionine, and
cystine withdrawn and replaced
by ammonium sulfate
Growth was observed in cultures of Claviceps purpurea
incubated in $00 ml. Erlenmeyer flasks on $0 ml. of
• - • ........... - _ ' " "s
medium and shaken at room temperature at 80 cycles/min.
slow
good
rapid
rapid
rapid
rapid
rapid
Dried myeelia grown on all these different media
were analyzed for ergothioneine by chromatographing
! extracts on alumina columns. The results are shown in
| Figures £-7. Ergothioneine was found to be present in
| each case. It therefore appears that the fungus does
| not require an organic source of sulfur for ergothio
neine biosynthesis. Furthermore, since ergothioneine
I was present in the absence of an external source of
| histidine, the fungus does not require a preformed
! imidazole ring for ergothioneine biosynthesis.
| It was possible, after fungal adaptation, to cul-
| ture Claviceps on a small number of amino acids.
Satisfactory growth was obtained which approached but
did not equal that obtained on more complex nitrogen
sources previously described.
! The nutritional approach to the problem of ergothio-
i '
j neine biosynthesis was predicated on discovering a medium
which would permit reasonably rapid growth, but in which
i . . . . . . .
1 ergothioneine biosynthesis would be reduced to a very
low rate. Supplementation of such a medium with pre
sumed precursors could then supply information on the
pathway of ergothioneine biosynthesis. On the basis
of the results reported above, it appears that Claviceps
is able to synthesize ergothioneine rapidly from simple
Figure 5
ALUMINA CHROMATOGRAPHY OF CLAVICEPS PURPUREA
GROWN ON ACID HYDROLYZED CASEIN AS THE
PRINCIPAL NITROGEN SOURCE
Klett
Units
100“
90"
80“
70“
60"
Ergo
thioneine
Histidine
10
0 5 10 15 20 25
ml. column effluent i
J
A four ml. aliquot of 10 ml. of fungal extract (prepared |
j
by refluxing 0.85 gm. of dried fungus with 10 ml. of 75$
. i
!
aqueous ethanol for 30 min.) was placed on the column and j
eluted with 75$ aqueous ethanol containing 1$ formic acid j
at the flow rate of 1+ ml. per hour. Five ml. fractions j
were collected. One ml. aliquots of each fraction were !
(21) |
taken to dryness and analyzed by the Melville-Lubschez testi
Figure 6
ALUMINA CHROMATOGRAPHY OF CLAVICEPS PURPUREA
GROWN ON A SYNTHETIC AMINO ACID MIXTURE AS
THE PRINCIPAL NITROGEN SOURCE
Klett
Units
Ergo
thioneine
o 5 io 15 20 25 30 35 l+o J+5 5o 55
ml. column effluent
A four ml. aliquot of 10 ml. of fungal extract (prepared
by refluxing 0.85 gm. of dried fungus with 10 ml. of 75$
aqueous ethanol for 30 rain.) was placed on the column and
eluted with 75$ aqueous ethanol containing 1$ formic acid
at the flow rate of I 4 . ml. per hour. Five ml. fractions
were collected. One ml. aliquots of each fraction
taken to dryness and analyzed by the Me 1 v I lie - Lub s che z jtest
50
Figure 7
ALUMINA CHROMATOGRAPHY OF CLAVICEPS PURPUREA
GROWN ON A SYNTHETIC AMINO ACID MIXTURE WITH
HISTIDINE, METHIONINE, AND CYSTINE ABSENT
AND REPLACED WITH AMMONIUM SULFATE
Klett
Units
80'
Ergo
thioneine
50
ko
Histidine
20"
10
0 5 10 15 20 25 30 35 l+o 1+5 5o 55
ml. column effluent
A four ml, aliquot of 10 ml. of fungal extract (prepared
by refluxing 0.85 ©»• of dried fungus with 10 ml. of 75$
aqueous ethanol for 30 min.) was placed on the column and
eluted with 75$ aqueous ethanol containing 1% formic acid
at the flow rate of 1+ ml, per hour. Five ml. fractions
were collected. One ml. aliquots of each fraction were
taken to dryness and analyzed by the Melville-Lubschez
(21) procedure.
51
carbon and sulfur sources, so that a nutritional
approach would not be successful unless ergothioneine
biosynthesis can be made limiting by addition of an
inhibitor or by the production of ergothioneine mutants.
Histidine Uptake
A Claviceps culture was incubated in a raannitol-
histidlne medium and aliquots were removed at hourly
Intervals and analyzed for histidine. The method is
described in more detail on page 26.
The values obtained for the histidine remaining
in the medium steadily decreased until a value of approx
imately 50% of the original value was obtained within
8-10 hours of incubation. This value did not decrease
with longer periods of ineubation. These results are
shown in Figure 8 and suggest a stereospecific uptake
of histidine from the medium, presumably the L-form.
The experiment was therefore repeated using only
the L-enantiomer instead of the racemic mixture used
previously. Now Instead of a steady decrease leveling
off sharply when $0% of the compound had disappeared,
it was found that the L-histidine in the medium contin
ued to fall until it could no longer be detected.
Histidine uptake by Claviceps was studied because
Figure Q
HISTIDINE DISAPPEARANCE IN A CLAVICEPS CULTURE
2000
Mierograms ^50
of Histidine 1500
Per Ml. 1250
1000
75>0
250
2 k 6 8 10 12 ll| 16
Hours
Ten grams of fungus in 10 ml. of a 2% mannitol
solution containing 2500 micrograms of DL-histidin©
per ml. Aliquots of 0.02 ml. were withdrawn every 2
hours for analysis. The histidine was determined by
the Macpherson modification of the Pauly reaction and
read in the Klett with a No. 5U green filter.
53
of the obvious chemical and possible metabolic relation
ship of histidine to ergothioneine. Information was
particularly desired as to the rate of histidine uptake
and whether histidine lack influences the fungal content
of ergothioneine. The results indicate a rapid stereo-
specific uptake of L-histidine from the medium by Clavi
ceps. The possibility that histidine was catabolized
outside the cell to products failing to give the Mae-
pherson color reaction cannot be excluded. This should
be answered unequivocally when a radioactive histidine
uptake study is made. In the case of histidine and
presumably other amino acids this fungus lacks the
ability to assimilate both enantiomers from a racemic
mixture. This must be taken into consideration in
radioactive studies where a labeled substrate is present
in the external medium as a racemate. Its effective
concentration may be only one-half of its actual concen
tration. The stereospecificity of the fungus also opens
the question of the Influence of the D-isomer on the
uptake of its enantioraer. If this is a competetive
phenomenon the rate of uptake of an enantiomer would be
expected to be increased by the absence of its antipode.
It also presents for consideration the property of
stereoisomerism in any contemplated inhibitor studies.
5 k
The rate and rapidity with which Claviceps can
apparently withdraw histidine from the medium lend
further support to its suitability for future studies
employing this suspected precursor in ergothioneine
biosynthesis*
growth Studies
Growth was measured from the dry weight of fungus
obtained at different time intervals. Growth on 5 ml*
of medium yielded a maximum dry weight which was too
small to provide enough ergothioneine for analysis*
However growth on 10 ml. of medium produced a maximum
dry weight of 167 mg. The growth expressed as dry
weight of fungus obtained on 10 ml. of medium is shown
in Figure 9« If 0.1$ of the dry weight of the fungus
Is assumed to be ergothioneine, then 167 micrograms of
ergothioneine were synthesized. This amount is conven
iently fractionated on the alumina columns of the size
now employed.
Except for the estimates of ergothioneine content
shown In Figures 5>-7» no attempt has been made to
correlate quantitatively the rates of ergothioneine
synthesis and growth.
The small volume of 10 ml. of culture medium used
Figure 9
55
GROWTH CURVE OF CLAVICEPS PURPUREA
200 '
190
180
Hours
120
Growth obtained on 10 ml* of medium as described;
shake cultures were maintained^ at the rate of 80
cycles per minute at room temperature
56
for the rapid biosynthesis of an amount of ergothio
neine which is conveniently fractionated makes it
possible to maintain a high concentration of radio
isotope without using much material. Since histidine
at an initial concentration of 2.f> mg. per ml. rapidly
disappears from the medium within a 90 hour interval,
conditions appear favorable for a study of ergothio
neine biosynthesis using radio-histidine.
Chapter V
SUMMARY AND CONCLUSION
An attempt was made to seleet a biological system
in which ergothioneine biosynthesis could be shown to
occur. A preliminary investigation was made which in
cluded the rat, rabbit, and the ergot fungus, Claviceps
purpurea.
The ergothioneine blood levels of the very young
rat and of the adult were compared and the fact verified
that the infant rat has no detectable blood ergothio
neine while adult rats were found to possess blood ergo
thioneine levels of 6 mg. per 100 ml. of blood. The
blood ergothioneine level of a rabbit was found to be
1* mg. per 100 ml. of blood. Using rabbit blood it was
established that ergothioneine is already present in
the reticulocyte stage at the same level as in the ma
ture erythrocyte.
It was discovered, using paper and alumina chroma
tography, that Claviceps purpurea, grown under controlled
laboratory conditions, could synthesize ergothioneine
from a medium containing a mixture of inorganic salts,
mannitol and an enzymic digest of casein. The relative
ly high content of approximately 1 mg. of ergothioneine
per gram of dry weight of fungus was found, which is
in fairly close agreement with the amount reported in
ergot sclerotia occurring naturally.
The relationship of the nutrition of the fungus to
ergothioneine production has been investigated in a
qualitative manner. Radical changes in the amino acid
components of the medium resulted in poor growth.
However it was found that the number of amino acids in
the culture medium could be markedly reduced with only
moderate decrease in growth rate.
It has been established that on a simplified medium,
with methionine, cystine, and histidine absent, ergo
thioneine biosynthesis can occur without an organic
source of sulfur or a preformed Imidazole ring.
The uptake of histidine, a suspected precursor of
ergothioneine, has been found to be rapid and stereo-
specifie for the L-enantiomer.
The growth of the fungus has been measured as a
function of time. Small flasks were used, in contem
plation of future work with radioisotopes, for the pur
pose of maintaining a small amount of radioisotope in
high concentration.
The work has demonstrated that ergothioneine bio
synthesis cannot readily be made limiting by nutrtional
59
means, and Information has been gathered in preparation
for a study of the synthesis utilizing tracer techniques.
\
\
\
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University o f Southern CcNtem ta Ll&rory
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Asset Metadata

Creator Ban, Robert William (author)

Core Title Studies on ergothioneine

School Department of Biochemistry

Degree Master of Science

Degree Program Biochemistry

Degree Conferral Date 1956-01

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-778100

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