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Isolation, purification and characterization of N5-formyltetrahydrofolic acid (folinic acid) from the housefly
Life Sciences Vol. 4, pp . 1573-1580, 1985 . Great Britain .
Per~mon Press Ltd.
Printed in
ISOIATION, PUTtIFICATIOH Al® CAaaAf~~l2em~rpA OF p5-
LIC BCID (P"OISHIC I1CID.) . FBOM THE HOiT88FLY Steve Miller and Albert S. Percy
Biology~Chemi.etry Section, Technology Hraach, r1 ~n+i cable nf.sease Center, Pablic Health Service, ü. S. Department of Health, Education, sad Welfare, Savannah, Georgia
(Received 31 May 1985) Folic acid coeazymea have been isolated from various sources {1,2,3,4) .
In the
housefly fo]3c acid is converted to a compound at the tetrahydro level duriag~ the papal stage of deveiopment
(5) "
Aa s result of this conversion the hovse-
ily upon emergence has a reserve tetrahydrofolic acid pool .
Ia the presence
of folic acid aatagoaists such es ami.aopterin or smethapteria growth eaà. development of the housefly larvae ie largely suppressed (5 ) " This paper is the first study undertaken for the isolation sad purification of a folic acid derivative from an insect and the peper describes new methods for such a purification .
These methods should be applicable for the purification
of folate derivatives from other sources. Materials and Methods IBrvae of the comsoon housefly Muaca domestics (L) were reared on a chemically defined diet under strictly sterile conditions .
The diet consisted
of vitamin flree casein, vitamins, salts end cholesterol in as agar base medium . F],y eggs were surface sterilized with 10;d neutral formalia before dispensing aseptical],y into the autoclaved medium .
A detailed description of the method
is given elsewhere (5) . For bacterial assq the three test organi.a®s used were Lactobsci»+," oerei, (ATCC
7469),
3treptococcue faecalis (ATCC
1573
8043)
sad Pediococcus cerevisae
1574
FOLIIdIC ACm FROM THE HOUSEFLY
(ATCC 8081) .
Vol. 4, No . 18
The methods of sassy and growth medium have been described (6,7) .
!blink acid (dl-AS -formyltetrabydrofolic acid) was used es a standard, and was flu~nished by Eli Lilly Research laboratories .
+t
Paper ohromatography sad bioautography wes carried out by conventional procedures using Vhatman Fo, 1 filter paper and descending techniques . vente for the paper chromatographic separations consisted of : water (3 :2 whims for whims) ; volume);
The sol-
I. 2,6-lutidine-
II . pyridine-propanol-water (1 :1 :1 whims for
III. ethanol-isobutyl alcohol-water-ammonium hydroxide (5 :3 :15 :0,3
whims for volume);
IP. Propanol-ethanol-ammonium hydroxide-water (10 :20 :1,25 :10
whims fas volume) (Table 1) . Purification, Procedures Preparation of Homogenate .
Homogenates were prepared by triturating 30
grams of flies in a mortar with 180 ml of .05 M sodium phosphate buffer pH 7.5 followed by homogenization in a Potter-Elvehjen glees hoa~ogeaizer .
(moss par-
tides were then removed by filtering through surgical cotton gauze. 0
haamgenate was autoclaved at 120 tore .
The
for twenty minutes and cooled to roam temperar
This vas follaved by ceatritligation at 20,000 x g for tea minutes end the
supernatant liquid vas decanted .
The supernatant liquid vas assayed for folgte
activity with P. cerevisae and reduced to a volume of 25 ml in vacuo (Rotovap, bath t emperature about 45° ) .
A11 operations were carried out under dim illumi-
nation. Malryeis .
The 25 ml concentrate was then dialyzed is a refrigerator
against Bauble distilled water with change of the dialyzing solution every 12 àrntra for a period of 2 days .
The dialyzable material vas then combined, and the
solvent vas removed in vacuo .
The resulting concentrate vas brought to a specific
volume with voter for the purpose of assaying with P. cerevisee.
The voter wan
removed iram the dialyzable material in vacuo sad the aoa-dialyzable material was discarded. BAS chromato~a~.
Column chromatography vas performed on T~ cellulose
* Trade names are for identification purposes sad do sot constitute endorsement by the Public Health Service.
Yol. 4, No . 18
FOLIIJIC ACm FROM THE HOUSEFLY
with an exchange capacity of .68 m equiv~gram.
1575
Zhe D$AE cellulose xsa washed
with .25 M NaOH until the washings were colorless .
This ras follored by noshing
with water until the pH of the suspension vas approximately 7.
A slurry of the
adsorbent is refer yes poured into a 2.5 x 30 cm glass column nod. allowed to settle .
After 200 ml of eater had passed through the column, the column ras
alloyed to drain to incipient dryness.
The dialysable material vas dissolved in
.5 ml water and then yes layered caref+~lltiy on top o! the column . eluted rith .05 M phosphate butter pH 7 .5 " ml fractions .
The column was
The elutes vas collected in five 50
All fractions displaying P. cerevisae activities were caaibined and
then concentrated to dryness in vacuo for t1uthen purification .
Attea~ts vete then
made to desalt the ca®bined tY~action before attesting ttu~ther purification . This vas accca~lished by adding 20 ml of 20~ ethanol to the ca®bined dry active fraction
n~ the DEAS column and ceatrifugi.ng under refrigerstioa .
The supnrnataae liquid .
vas decanted into a flask, assayed, and then evaporated to dryness user vacno. The elution patterns of the purified folic acid derivatives cram the housafltiy sad foliaic acid-S[r ere shown in Rig. 1. Cellulose pander chromatography .
Twenty grams of lmatmsa cellulose porder
xaa mixed with 150 ml of the solvent propaaol :etheaol :vater amoonium bydrwdde (10 :5 :30 : .5 volume for volume) sad. vigorously stirred.
The adxture van then
poured into a column (2 .5 cm x 3o cm) and alloyed to settle . by washing the coltam3 with 200 ml of the above solvent .
This vas follwed
After the wash the column
was allAVed to drain to dryness and pacün8 of the column ras completed by appllr ing pressure at both ends of the column with steel rods .
The active material,
previouslsr treated with ~ ethanol, vas dissolved in .g al rater and then the material was caretY~llry introduced into the column and eluted with the above solvent system .
Zones, es vieyed by ultraviolet light were collected as fractions in flasks .
The solvent in the flasks was then removed under vacuo sad twenty ml of ester vas added to each flask.
E~ch fraction was seared rith P. cerevisae and then the
eater was removed ~oai the active material .
The cellulose ponder column served
for the ttu~ther removal of salts sad the separation of a material that had no folate activity .
1576
FOLINYC ACm FROM THE HOUSEFLY
Yol. 4, No. 18
TABLE l Paper (ätra®sto~sphic Ooo~arisoa of Authentic Folinic Acid with s àblic Acid Derivative Isolated trom Sousetlies .
Bolvent B~ste~m I II III 111
Folic Acid Derivative .I+O .24
Folinic /cid-SF .~1 .24
.24 .09
.26 .09
_o á
FIG.1 EUJTION WRTERNS OF AUTFENTIC FOLOIIC ACID :- . , AND FOLIC ACID DERII~IVE (FLf EXTRACTI :-___ ~i~9 OF AUTNENTID FDLINIG ADID AND I6 /~.~ Of FOLID ADID DERIVATIYé IEAS ADOLD iL'A1IATELY TO DEAL OOLUNINi . TNE ¢1/~IIT 1EAi/~: fIW"f11ATL WffER . 60 YL fRAOT10N" WERE CDLLS.TED. ACTIVITIEE WERE YEAlIURED YÍITN rEö7Ö=
COCGUf QENENSAE.
Vol. 4, No . 18
FOLIIIIC AC>D FROM THE HOUSEFLY
Thin layer chromatogreçpby.
1577
Tlro glass plates 20 x 20 cm were laden with a
rapidly stirred cellulose ponder mixture (30 gams of cellulose in ~+5 ml of water) in a lawyer 60 microns in thickaese using an applicator .
The plates were dried in
an oven for one hour at 1050 . Results Ia the puriricatioa procedures autoclaving under sligbtty a7.kaline coaditions follaved by centrifugation re®oved gross particles from the homogenate . Eighty micrograms of folerte activity vas p~reaent is the centrifuged homogenate . Malysie effected tlu~ther purirication and, it xaa ehova by assay that at least 80¢ of the folgte activity (60 micrograms) vas present is the dislyzable fractions. Further purification was effected by IEAS chromatography .
After all fractions
were collected from the IEAS colamm, colored material remained on the columm sad the 4actione containing no tolate activity also were colored. the effectiveness of the column in purification .
This demonstrated
The combined active fraction item
this column contained approximately 9~+ of the active material applied (55 microgreme), however the material vas not sufficiently pure for ultraviolet determination. Following the re~ova]. of salts es described under Methods, t~rther purirication vas attempted by column chremstogaphy using Florieil sad starch as aàsorbants .
Dliatinct separations of compounds did not occur on such co].umms .
It vas
then thought that separations of caapounds at this stage of purification involved small differences is Rf values thus making column chromatogr~p~y uasuccesaful as a tool for purification . puriricatian .
This vas due to the limited amount of active material that can be
e8plied to the paper . factory.
Paper chromatogapby could not be used as a method of
Also, recovery of such material lY~on paper vas not satis-
Further separations of compounds were brought about by thin ].gyer
chromatogre~y.
Tw plates were then spotted with 20 micrograms of folgte activ-
ity from the cellulose ponder column dissolved in 0 .2 ml of water.
Alter appli-
cation, the plates were air dried for one hour and then placed ín a far psevious]y equilibrated with the solve. vas 2,6-lutidine-water (3 :2
The solvent system Sór the first thin le`yer plates
vw) .
The development time was gpprorimste7,T three
1578 hours.
FOLIIQIC ACm FROM THE HOUSEFLY
Vol. 4, No. 16
The platen were then removed and dried in air. Three distíact $ones Were visible under ultraviolet light .
Starting from
the origin there Was a blue rivoreacefft zana, lolloved by a brown zone and then s brilliant green fluorescent zone .
Each oí the zones Was caretlL7.ly geparatcd tram
the other zone with a spatula sad these areas Were scrag~ed from the plate into 50 ml beakers .
Twenty al of Water vsa added to each beaker .
After 16 hours in the
refrigerator the beakers Were stirred and the material in the beakers Was centriwed to remove the cellulose powder,
The liquid tram the ceatritUge tubes Was
decanted into flanks, assayed and Water Was removed from the riask containing the active material .
This Was the procedure used for each subsequent elution oí com-
pounds tram chin layer chraoatograms.
The brown zone contained 18 micrograms oí
tolace activity and this material Was dissolved is .2 ml water then applied to two thin ].,yer plates . The solvent system for the second thin ].ever chr~matogram wds propaaol : ethenol :vater :ammonium Y~ydro~de (1 :5 :3 : .05 were visible under ultraviolet light .
vw) .
After development tour zones
Starting prom the origin, there was an
orange, a blue riuorescent, a brown, ~ a green riuorescent zoo, zone contained 16 micrograms oí folste activity .
The brown
The 16 micrograms oí tolste
activity dissolved in 0.2 ml water Was then applied to tw thin layer plates . The solvent system for the third thin layer chromatogram was propanol :Water : 2,6-lutidine (l :1
a vw) .
ultraviolet light.
Three zones were visible on the chromatogram under
Starting tram the origin, s blue rivoresce~, a nontluores-
teat sad s green riuorescent zone . grams of folate activity .
The nonriuoresce~ zone contained 14 micro-
An ultraviolet spectrum oí this active material wen
determined with s Cory Model 14 Spectrophotometer. A schematic outline for the purification oí the Colic acid derivative from housenies is given in Fig. 2. The ultraviolet peak absorption oí this folic acid derivative at 287 mu is identical With authentic tolinic acid .
The folic acid derivative shoved no
shift in spectrum after tlu~ther ~ and thin le~yer chromatography .
Only one
spot e~ppeared oa the bioautograph when the derivative wen chromatographed alone
Vol. 4, No. 18
FOLINIC ACID FROM THE HOUSEFLY
1579
8oaogaoaa 1. Autoclaw Omtrült®a
12.
ï'-'
8u~aeoatsot
Ppt. diaaard)
Ma7,ysia 0° varaus vatar
1 nLal~sabla (85})
liOndialysabla 15~ (diacard)
~~ chrorato~r lonactin lractiaos
ooüaad act~n iraatiaw 1, ithaool tsaatamt ~. ~t~tngt
egparoatant
Basidue (diaoarà)
Gllulosa poYdar Q~raaato~r 3awatin soon (diaoarà) ero~o saw
datin saw 1st ~ 14w oLs+aststoR'ea
Blw fluorasoant sow
~~ tlncayacaat sow
9chmatic outliaa !br the aapantioa of a tolk acid dalwoon tras tàe honnlly
or coohiromatogreyphed with folinic acid-SF.
Both the isolated derivative cad the
autàentic caarpoimd migrated with essentially the sane R! vaTuea in lour solvent syete~s.
~e lblic acid derivative vas oleo active for all three microorgaaiens
L. celei . P. cerevisae, end 3. laecalie .
Fines
the derivative bald the sa®e
elution pattern u folinic acid-SF on a ~A8 column. ~erefore this weight of evidence indicates that the folic acid derivative ie ~-forrgyltetrahydrofolic acid (folinic acid) .
Folic acid coenzymes in tha
housefly are currently under ttu~ther investigation .
1580
FOLIIdIC ACm FROM THE HOUSEFLY
Vol. 4, No. 18
Methods are presented for the isolation and purification of H5-for~ltetrar ~yydrofolic acid t5ron the housefly .
By the coa~limentary effect of BAS column
chromatography and repeated thin layer chrc®atogra~y the folate derivative vas sufficiently pt~ified to give an ultraviolet apactr~ ide~ical with authentic tb].inic acid .
Other characteristics presented are also si~eilar . Aokaov].ed8es~eat
~e mithoz~s rash to thank Mrs. Jeanette Oollins !br her excellent tachaical assistance . References l.
J. M. 1~OI~SA sad M. sILVSßMAF, J. Biol . Chew. ?~, 3299 (1962) .
2.
J. B. iïrJ.'i~G, J. M. I+AORO1CíA sad M. sILVSßMAF, Biochem. J. 8~, 9 (1962) .
3.
s. USßIIi, J. Biol . Chew . 2,~~+, ?373 (1959) "
4.
B. iIItIGST, J. Amer . Chew. soc .
5.
A.
6.
s. s. S1C4LL, B. M. GUIItARD sad R. J. üIIT .r~, J. Biol . Chew. 14~, 519 (192) .
~.
L. M. FL7~i, V. B. itiLT3~, B. L. 0' ~Ii~ sad A» G. HOGAA, Anal . Chew. ~0,
Tj, 3930 (1955) "
s. PSRRY and s. MIIJA$t, J. of Insect Pliysiolo®, in press.
730 (1951) .
Per~mon Press Ltd.
Printed in
ISOIATION, PUTtIFICATIOH Al® CAaaAf~~l2em~rpA OF p5-
LIC BCID (P"OISHIC I1CID.) . FBOM THE HOiT88FLY Steve Miller and Albert S. Percy
Biology~Chemi.etry Section, Technology Hraach, r1 ~n+i cable nf.sease Center, Pablic Health Service, ü. S. Department of Health, Education, sad Welfare, Savannah, Georgia
(Received 31 May 1985) Folic acid coeazymea have been isolated from various sources {1,2,3,4) .
In the
housefly fo]3c acid is converted to a compound at the tetrahydro level duriag~ the papal stage of deveiopment
(5) "
Aa s result of this conversion the hovse-
ily upon emergence has a reserve tetrahydrofolic acid pool .
Ia the presence
of folic acid aatagoaists such es ami.aopterin or smethapteria growth eaà. development of the housefly larvae ie largely suppressed (5 ) " This paper is the first study undertaken for the isolation sad purification of a folic acid derivative from an insect and the peper describes new methods for such a purification .
These methods should be applicable for the purification
of folate derivatives from other sources. Materials and Methods IBrvae of the comsoon housefly Muaca domestics (L) were reared on a chemically defined diet under strictly sterile conditions .
The diet consisted
of vitamin flree casein, vitamins, salts end cholesterol in as agar base medium . F],y eggs were surface sterilized with 10;d neutral formalia before dispensing aseptical],y into the autoclaved medium .
A detailed description of the method
is given elsewhere (5) . For bacterial assq the three test organi.a®s used were Lactobsci»+," oerei, (ATCC
7469),
3treptococcue faecalis (ATCC
1573
8043)
sad Pediococcus cerevisae
1574
FOLIIdIC ACm FROM THE HOUSEFLY
(ATCC 8081) .
Vol. 4, No . 18
The methods of sassy and growth medium have been described (6,7) .
!blink acid (dl-AS -formyltetrabydrofolic acid) was used es a standard, and was flu~nished by Eli Lilly Research laboratories .
+t
Paper ohromatography sad bioautography wes carried out by conventional procedures using Vhatman Fo, 1 filter paper and descending techniques . vente for the paper chromatographic separations consisted of : water (3 :2 whims for whims) ; volume);
The sol-
I. 2,6-lutidine-
II . pyridine-propanol-water (1 :1 :1 whims for
III. ethanol-isobutyl alcohol-water-ammonium hydroxide (5 :3 :15 :0,3
whims for volume);
IP. Propanol-ethanol-ammonium hydroxide-water (10 :20 :1,25 :10
whims fas volume) (Table 1) . Purification, Procedures Preparation of Homogenate .
Homogenates were prepared by triturating 30
grams of flies in a mortar with 180 ml of .05 M sodium phosphate buffer pH 7.5 followed by homogenization in a Potter-Elvehjen glees hoa~ogeaizer .
(moss par-
tides were then removed by filtering through surgical cotton gauze. 0
haamgenate was autoclaved at 120 tore .
The
for twenty minutes and cooled to roam temperar
This vas follaved by ceatritligation at 20,000 x g for tea minutes end the
supernatant liquid vas decanted .
The supernatant liquid vas assayed for folgte
activity with P. cerevisae and reduced to a volume of 25 ml in vacuo (Rotovap, bath t emperature about 45° ) .
A11 operations were carried out under dim illumi-
nation. Malryeis .
The 25 ml concentrate was then dialyzed is a refrigerator
against Bauble distilled water with change of the dialyzing solution every 12 àrntra for a period of 2 days .
The dialyzable material vas then combined, and the
solvent vas removed in vacuo .
The resulting concentrate vas brought to a specific
volume with voter for the purpose of assaying with P. cerevisee.
The voter wan
removed iram the dialyzable material in vacuo sad the aoa-dialyzable material was discarded. BAS chromato~a~.
Column chromatography vas performed on T~ cellulose
* Trade names are for identification purposes sad do sot constitute endorsement by the Public Health Service.
Yol. 4, No . 18
FOLIIJIC ACm FROM THE HOUSEFLY
with an exchange capacity of .68 m equiv~gram.
1575
Zhe D$AE cellulose xsa washed
with .25 M NaOH until the washings were colorless .
This ras follored by noshing
with water until the pH of the suspension vas approximately 7.
A slurry of the
adsorbent is refer yes poured into a 2.5 x 30 cm glass column nod. allowed to settle .
After 200 ml of eater had passed through the column, the column ras
alloyed to drain to incipient dryness.
The dialysable material vas dissolved in
.5 ml water and then yes layered caref+~lltiy on top o! the column . eluted rith .05 M phosphate butter pH 7 .5 " ml fractions .
The column was
The elutes vas collected in five 50
All fractions displaying P. cerevisae activities were caaibined and
then concentrated to dryness in vacuo for t1uthen purification .
Attea~ts vete then
made to desalt the ca®bined tY~action before attesting ttu~ther purification . This vas accca~lished by adding 20 ml of 20~ ethanol to the ca®bined dry active fraction
n~ the DEAS column and ceatrifugi.ng under refrigerstioa .
The supnrnataae liquid .
vas decanted into a flask, assayed, and then evaporated to dryness user vacno. The elution patterns of the purified folic acid derivatives cram the housafltiy sad foliaic acid-S[r ere shown in Rig. 1. Cellulose pander chromatography .
Twenty grams of lmatmsa cellulose porder
xaa mixed with 150 ml of the solvent propaaol :etheaol :vater amoonium bydrwdde (10 :5 :30 : .5 volume for volume) sad. vigorously stirred.
The adxture van then
poured into a column (2 .5 cm x 3o cm) and alloyed to settle . by washing the coltam3 with 200 ml of the above solvent .
This vas follwed
After the wash the column
was allAVed to drain to dryness and pacün8 of the column ras completed by appllr ing pressure at both ends of the column with steel rods .
The active material,
previouslsr treated with ~ ethanol, vas dissolved in .g al rater and then the material was caretY~llry introduced into the column and eluted with the above solvent system .
Zones, es vieyed by ultraviolet light were collected as fractions in flasks .
The solvent in the flasks was then removed under vacuo sad twenty ml of ester vas added to each flask.
E~ch fraction was seared rith P. cerevisae and then the
eater was removed ~oai the active material .
The cellulose ponder column served
for the ttu~ther removal of salts sad the separation of a material that had no folate activity .
1576
FOLINYC ACm FROM THE HOUSEFLY
Yol. 4, No. 18
TABLE l Paper (ätra®sto~sphic Ooo~arisoa of Authentic Folinic Acid with s àblic Acid Derivative Isolated trom Sousetlies .
Bolvent B~ste~m I II III 111
Folic Acid Derivative .I+O .24
Folinic /cid-SF .~1 .24
.24 .09
.26 .09
_o á
FIG.1 EUJTION WRTERNS OF AUTFENTIC FOLOIIC ACID :- . , AND FOLIC ACID DERII~IVE (FLf EXTRACTI :-___ ~i~9 OF AUTNENTID FDLINIG ADID AND I6 /~.~ Of FOLID ADID DERIVATIYé IEAS ADOLD iL'A1IATELY TO DEAL OOLUNINi . TNE ¢1/~IIT 1EAi/~: fIW"f11ATL WffER . 60 YL fRAOT10N" WERE CDLLS.TED. ACTIVITIEE WERE YEAlIURED YÍITN rEö7Ö=
COCGUf QENENSAE.
Vol. 4, No . 18
FOLIIIIC AC>D FROM THE HOUSEFLY
Thin layer chromatogreçpby.
1577
Tlro glass plates 20 x 20 cm were laden with a
rapidly stirred cellulose ponder mixture (30 gams of cellulose in ~+5 ml of water) in a lawyer 60 microns in thickaese using an applicator .
The plates were dried in
an oven for one hour at 1050 . Results Ia the puriricatioa procedures autoclaving under sligbtty a7.kaline coaditions follaved by centrifugation re®oved gross particles from the homogenate . Eighty micrograms of folerte activity vas p~reaent is the centrifuged homogenate . Malysie effected tlu~ther purirication and, it xaa ehova by assay that at least 80¢ of the folgte activity (60 micrograms) vas present is the dislyzable fractions. Further purification was effected by IEAS chromatography .
After all fractions
were collected from the IEAS colamm, colored material remained on the columm sad the 4actione containing no tolate activity also were colored. the effectiveness of the column in purification .
This demonstrated
The combined active fraction item
this column contained approximately 9~+ of the active material applied (55 microgreme), however the material vas not sufficiently pure for ultraviolet determination. Following the re~ova]. of salts es described under Methods, t~rther purirication vas attempted by column chremstogaphy using Florieil sad starch as aàsorbants .
Dliatinct separations of compounds did not occur on such co].umms .
It vas
then thought that separations of caapounds at this stage of purification involved small differences is Rf values thus making column chromatogr~p~y uasuccesaful as a tool for purification . puriricatian .
This vas due to the limited amount of active material that can be
e8plied to the paper . factory.
Paper chromatogapby could not be used as a method of
Also, recovery of such material lY~on paper vas not satis-
Further separations of compounds were brought about by thin ].gyer
chromatogre~y.
Tw plates were then spotted with 20 micrograms of folgte activ-
ity from the cellulose ponder column dissolved in 0 .2 ml of water.
Alter appli-
cation, the plates were air dried for one hour and then placed ín a far psevious]y equilibrated with the solve. vas 2,6-lutidine-water (3 :2
The solvent system Sór the first thin le`yer plates
vw) .
The development time was gpprorimste7,T three
1578 hours.
FOLIIQIC ACm FROM THE HOUSEFLY
Vol. 4, No. 16
The platen were then removed and dried in air. Three distíact $ones Were visible under ultraviolet light .
Starting from
the origin there Was a blue rivoreacefft zana, lolloved by a brown zone and then s brilliant green fluorescent zone .
Each oí the zones Was caretlL7.ly geparatcd tram
the other zone with a spatula sad these areas Were scrag~ed from the plate into 50 ml beakers .
Twenty al of Water vsa added to each beaker .
After 16 hours in the
refrigerator the beakers Were stirred and the material in the beakers Was centriwed to remove the cellulose powder,
The liquid tram the ceatritUge tubes Was
decanted into flanks, assayed and Water Was removed from the riask containing the active material .
This Was the procedure used for each subsequent elution oí com-
pounds tram chin layer chraoatograms.
The brown zone contained 18 micrograms oí
tolace activity and this material Was dissolved is .2 ml water then applied to two thin ].,yer plates . The solvent system for the second thin ].ever chr~matogram wds propaaol : ethenol :vater :ammonium Y~ydro~de (1 :5 :3 : .05 were visible under ultraviolet light .
vw) .
After development tour zones
Starting prom the origin, there was an
orange, a blue riuorescent, a brown, ~ a green riuorescent zoo, zone contained 16 micrograms oí folste activity .
The brown
The 16 micrograms oí tolste
activity dissolved in 0.2 ml water Was then applied to tw thin layer plates . The solvent system for the third thin layer chromatogram was propanol :Water : 2,6-lutidine (l :1
a vw) .
ultraviolet light.
Three zones were visible on the chromatogram under
Starting tram the origin, s blue rivoresce~, a nontluores-
teat sad s green riuorescent zone . grams of folate activity .
The nonriuoresce~ zone contained 14 micro-
An ultraviolet spectrum oí this active material wen
determined with s Cory Model 14 Spectrophotometer. A schematic outline for the purification oí the Colic acid derivative from housenies is given in Fig. 2. The ultraviolet peak absorption oí this folic acid derivative at 287 mu is identical With authentic tolinic acid .
The folic acid derivative shoved no
shift in spectrum after tlu~ther ~ and thin le~yer chromatography .
Only one
spot e~ppeared oa the bioautograph when the derivative wen chromatographed alone
Vol. 4, No. 18
FOLINIC ACID FROM THE HOUSEFLY
1579
8oaogaoaa 1. Autoclaw Omtrült®a
12.
ï'-'
8u~aeoatsot
Ppt. diaaard)
Ma7,ysia 0° varaus vatar
1 nLal~sabla (85})
liOndialysabla 15~ (diacard)
~~ chrorato~r lonactin lractiaos
ooüaad act~n iraatiaw 1, ithaool tsaatamt ~. ~t~tngt
egparoatant
Basidue (diaoarà)
Gllulosa poYdar Q~raaato~r 3awatin soon (diaoarà) ero~o saw
datin saw 1st ~ 14w oLs+aststoR'ea
Blw fluorasoant sow
~~ tlncayacaat sow
9chmatic outliaa !br the aapantioa of a tolk acid dalwoon tras tàe honnlly
or coohiromatogreyphed with folinic acid-SF.
Both the isolated derivative cad the
autàentic caarpoimd migrated with essentially the sane R! vaTuea in lour solvent syete~s.
~e lblic acid derivative vas oleo active for all three microorgaaiens
L. celei . P. cerevisae, end 3. laecalie .
Fines
the derivative bald the sa®e
elution pattern u folinic acid-SF on a ~A8 column. ~erefore this weight of evidence indicates that the folic acid derivative ie ~-forrgyltetrahydrofolic acid (folinic acid) .
Folic acid coenzymes in tha
housefly are currently under ttu~ther investigation .
1580
FOLIIdIC ACm FROM THE HOUSEFLY
Vol. 4, No. 18
Methods are presented for the isolation and purification of H5-for~ltetrar ~yydrofolic acid t5ron the housefly .
By the coa~limentary effect of BAS column
chromatography and repeated thin layer chrc®atogra~y the folate derivative vas sufficiently pt~ified to give an ultraviolet apactr~ ide~ical with authentic tb].inic acid .
Other characteristics presented are also si~eilar . Aokaov].ed8es~eat
~e mithoz~s rash to thank Mrs. Jeanette Oollins !br her excellent tachaical assistance . References l.
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