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The inability of thiols to reduce cobalamins in the absence of a metalion
Volume 1, number 5
FEBS LETTERS
Oct0b¢i 19fi8- "
THE I1NAB]L]TY O F T m O L S TO REDUCE COBALAMIiNS IN THE ABSENCE O F A METAL ION * D.CAVALIM~, R.SCANDURRA, B.BARBONI and M~MARCUCCiZ n s t i t u t e Of BiOlo~cal Chemistry o f the o f Roma, ~t,'Jy
U,';h,e.~
Rec~ive_.d 1B Septeanbvx t96B
tt has been reported ~hat cysteine, thioglycolic acid and other thiols are able to reduce cobalamins |1--3]. Since reduced cobalamins are reo~dized by molecular oxygen, coba]amins are able therefore to catalyze the oxidation o f thiols in the presence o f air [4]. We have found, however, that the reduction o f h y d m x o c o b a l amin (B12a) by thiols is indirect and is mediated b y a metal ion present as an hnpurity in the B~2a sam#eReduction o f BI2 a has been followed by recording the spectral curve in the range 2"20 dO0 m p b'..: ~ Beckman DK2A spectrophotometer. Quartz cuve~tes converted in Thunbcrg tubes, provided with 2 side arms, were used. ] n t h e c e n t r a l c a v i t y w a s p l a c e d 0 . 0 9 / . t m o ] e s
o f B l 2 a (Pierre]. Milano) dissolved in 2 nil H 2 0 and 0_5 mi 0.02 M acetate buffer pH 4.2. ]n one side arm was placed 0.45 pmo]es cysteamine hydrochloride dissolved in 0.3 m] H 2 0 . tn ~he other side arm was placed 0.2 mi o f 0.5 M glycine-NaOH buffer ~H 8.9. The tube was evacuated with a water p u m p and refilled with argon three times. Fig. t shows the spectral curve o f B12a at p]-] 4.2 - (aquocobalamin ;k max 350 nap). When cystearnine is added at pH 4.2 the curve Bt2 a changes to filet rep o r i e d f o r t h e c o m p l e x o=~B12a w i t h t h i o l s [ 3 # ] ~U n d e r
the preseni conditions the complex is f o r m e d s l o w l y and Ieaches its m a x i m m n hi 20 minutes. No reduction ~of B I za is o b ~ r v e d at pH 4.2. When the pH o f the solution containing the complex has risen from 4 2 t o g.9 by the addition ofglycin~NaOH buffer, Bi2a is re- ._ duced as seen b3r the slow change o f t~e spec _ t t ~ tO"- . that o f BI2 r [5]. T h e ~e,:iuction is a]m0st complete " m 60 lninutes. If an experiment ~hm'lar~o t h a t de.st:n'bed :': .
• siglio Nazionrdc de]l¢ Ricm'che. •
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1-0
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3 5 0 m p
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310 mJu
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40D
WAVELENGTH
20 rain after t h e a d d i t i o n o f cysteamine at pH 4.2; B12 z formation aftci increasingthe pH solution to 8.9; !: = after 2 mizi; 2 = afar4
~ ] n ; . 3 =. a f t e r 6 ~nin; 4 - - a f t e r 1 0 r a i n ; 5 = z_t'-
ter :20 rain; ~; = a . O e ~ 3 O
rain: 7-- after 45 rain; 8 -= after 60
mira "abov6is c a n i e d o n t !n:th e presence o f 2:5 X t 0 7 3 M •E ~ A , m £ c bmplcx:~bl-Bi~a at pH 422 is a g a~~ pr=~ fluced, :bui'the addifi on o f glycine-NaOH bnffer, does
Vo],rme 1, ~mntmx 5 -
F'JSBB LETT~_RS
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O~ober 1968
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35olm, ~
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0.1 270
200
290 300 WAVELE NGTH
350
270
400'
" 1 280
2 ~ 0 300 WAVELENGTH
350
40C
Fig. 3 . - - - - - B ] 2 a ~fter p ~ e througl~ a cheating resin, dissolved i n H 2 0 - -- . . . . . . . . . cystcamin= B12 a ( ~ th~augh
Fig= 2. ------B12a after passage through a chelating ze..s~, dissolved in H20. -- . . . . . . . cystezmine-Bl2a ( p z . . ~ through a chelating xesin) complex, 20 rain aftez the addition o f cy.'t~anine.
a chelating resin) compl¢, 20 rain z_qm the addition of'.he cystearr~ne. B 12;" .formation, a~1er additior, o f pP 8-9 buffer ~ the pjr~ence of C~++ 3 X lO -7 M. I ~- after 2 mi~; 2 = after 4 rain; 3 = after 6 rain; 4 = a~-ter 8 rain; 5 = after 10
- splitting of the cysteamine-B12a complex, aft¢~ ri.~ng the pH to 8=9; 1 = after 2 ~in; 2 = aftez 4 rain; 3 = afro': ] 0 rnin; 4 = after 20 rain; 5 = after 30 rain; 6 = after 60 rain.
nfin; 6 = afte~ 20 rain; 7 = z ~ e r 30 rain; 8 = m-to; 45 mir.; 9 =
after 60 ram.
s u m p t ~ e evidence for the necessity o f a metal ion im-
purity as an intermediate for the reduction o f E~2a, we
have eliminated eventual metals by passage o f ~3e
the reducticn o f B l 2 ~ b y cysteamJne has t o b e related with the removal o f a metal ion, the e x p e m a e n t zep o r t e d in fig. 2 has been repeated h-t the presence o f C'u++ i o n s As seen in fig. 3, the addition o f a trace o f Cu " ~ ions reintrofluce~ the reduction o f B]2 a b y c y s t e a m i n e . F e -~-~, Co - ~ , iNl-++ and Zn ++ have been assayed in the p]ace o f Cu ++, w i t h o u t ~LtCCe~. The ~ e c e s s i ~ for a metal ion has been observed also when
B ] 2 a solution through a chelating resin ( D o w e x A I in t h e Ha + form). Using the solution o f B 1 2 a passed through the chelating resin, t h e complex thiol-B12 a at p H 4.2 is again produced u p o n t h e addition o f c y ~ t e nmine (fig. 2); however, w h e n the p H o f Lhe solution is raised t o 8 . 9 no reduction occurs. In t h e place o f t h e c y s t e i n e o r m e r c a p t o e t h a n o l w e r e used i n t h e p l a c e curve o f Bl2r t h e curve is changrd ~ o w l y t o tlmt o f o f ¢ysteamme. BI2 a a t p H above 7. This observa~on ~ d l o a t e s t h a t the ]n t~e l ~ h t o f the ze.s~t~ reported above it apl~axs ~ t i o n in-the absence o f a rectal i o n produces- ' t h a t t h e reduction-of BI2 a b y th~ols is indirect..'ih =. t h e cleavage o f - t h e thi'oi-B12 a c o m v ; e ~ i n t h e p l a c e o f " presence o f a metal-ion is nece&~ry and a po~_~le exreducing BI2 a t o B l 2 r - I d e n t i c a l res~dts have been 0]3- * planation is t h a t t h e ffdol red~3ces the metal which in tahied w h e n Bt2a-fi'ee t r o m contamina~g.met~_i~ was • its turn ~ednces t h e c o b ~ l ~ m ~ n . T h a t t h i s is a reason.p r e p p e d using p.~per, elect~oph0resis in, 0.5. m acetic. _ -
"
a c i d. ] a 3 0 r d e r - t O e s t a b ~
"whether the
~uppress3on
of
-
27~
- ~olmme 1, ~umber 5
FEBS LE'rrERS
thai, u n d e r t h e conditions o f t h e l~esent expe~i~e~ts, C u + i n t h e form o f C a ~ disso]ved in 0.25 M KCTI actual]y reduces B12 a 1o B t 2 r in ~he ~ s e n c e o f a thiol at a Although t h e addition ©tz ~c.e o £ C ~ "H" ions m a y 1eplace t h e m e t ~ _f~moved b y the passage o f t h e B t 2 a solution through t h e r.~helating xes~n, we d o n o t so f~r ~:rave a n y evidence t o establish which metal is a c h ~ i l y present as an i m p m i t y in BI2 a cata]ysing t h e Teduction o f ~he coba'Jzmin b y thioIs.
274
O c ~ 0 ~ t96E
Refe~rences
~I] ~ ~ d R.Mu~ie, ] o ~ S~te L s ~ 26 (1952) s s s . [2] :].W_Ecabnoff,Bioahem. B i o p h ~ R~'~ Co~amun. 16 { 1 9 ~ ) 484. |3] M.Adler, T~edwi~k aha T.S~ozna~ki, J, Am. Chem. Soc. ZB (1966) 5 0 1 8 . " 14] Ji.Yo01, Bio~mm. J. 88 (1963) 295:, [~] E.A.Kackza, D.E.Wolf, F.~Kuehl Jz. and ICFolkers, Science 112 (1950) 354.
FEBS LETTERS
Oct0b¢i 19fi8- "
THE I1NAB]L]TY O F T m O L S TO REDUCE COBALAMIiNS IN THE ABSENCE O F A METAL ION * D.CAVALIM~, R.SCANDURRA, B.BARBONI and M~MARCUCCiZ n s t i t u t e Of BiOlo~cal Chemistry o f the o f Roma, ~t,'Jy
U,';h,e.~
Rec~ive_.d 1B Septeanbvx t96B
tt has been reported ~hat cysteine, thioglycolic acid and other thiols are able to reduce cobalamins |1--3]. Since reduced cobalamins are reo~dized by molecular oxygen, coba]amins are able therefore to catalyze the oxidation o f thiols in the presence o f air [4]. We have found, however, that the reduction o f h y d m x o c o b a l amin (B12a) by thiols is indirect and is mediated b y a metal ion present as an hnpurity in the B~2a sam#eReduction o f BI2 a has been followed by recording the spectral curve in the range 2"20 dO0 m p b'..: ~ Beckman DK2A spectrophotometer. Quartz cuve~tes converted in Thunbcrg tubes, provided with 2 side arms, were used. ] n t h e c e n t r a l c a v i t y w a s p l a c e d 0 . 0 9 / . t m o ] e s
o f B l 2 a (Pierre]. Milano) dissolved in 2 nil H 2 0 and 0_5 mi 0.02 M acetate buffer pH 4.2. ]n one side arm was placed 0.45 pmo]es cysteamine hydrochloride dissolved in 0.3 m] H 2 0 . tn ~he other side arm was placed 0.2 mi o f 0.5 M glycine-NaOH buffer ~H 8.9. The tube was evacuated with a water p u m p and refilled with argon three times. Fig. t shows the spectral curve o f B12a at p]-] 4.2 - (aquocobalamin ;k max 350 nap). When cystearnine is added at pH 4.2 the curve Bt2 a changes to filet rep o r i e d f o r t h e c o m p l e x o=~B12a w i t h t h i o l s [ 3 # ] ~U n d e r
the preseni conditions the complex is f o r m e d s l o w l y and Ieaches its m a x i m m n hi 20 minutes. No reduction ~of B I za is o b ~ r v e d at pH 4.2. When the pH o f the solution containing the complex has risen from 4 2 t o g.9 by the addition ofglycin~NaOH buffer, Bi2a is re- ._ duced as seen b3r the slow change o f t~e spec _ t t ~ tO"- . that o f BI2 r [5]. T h e ~e,:iuction is a]m0st complete " m 60 lninutes. If an experiment ~hm'lar~o t h a t de.st:n'bed :': .
• siglio Nazionrdc de]l¢ Ricm'che. •
.
.
.
_
1-0
•
a
•
3 5 0 m p
|
wt I1 I D ! |
310 mJu
"
>-
" ~ . ~ , .
z t
I # _ V
\
;
J
t
,
o
o.z
n.
0.5
m
",
"---.~'-~=~,-.._._..v-,
,,
<
".,.
,
:
0_1 I
270
~
1
.:280 . 2gO ,
-"
"|
....
300
1
:350
40D
WAVELENGTH
20 rain after t h e a d d i t i o n o f cysteamine at pH 4.2; B12 z formation aftci increasingthe pH solution to 8.9; !: = after 2 mizi; 2 = afar4
~ ] n ; . 3 =. a f t e r 6 ~nin; 4 - - a f t e r 1 0 r a i n ; 5 = z_t'-
ter :20 rain; ~; = a . O e ~ 3 O
rain: 7-- after 45 rain; 8 -= after 60
mira "abov6is c a n i e d o n t !n:th e presence o f 2:5 X t 0 7 3 M •E ~ A , m £ c bmplcx:~bl-Bi~a at pH 422 is a g a~~ pr=~ fluced, :bui'the addifi on o f glycine-NaOH bnffer, does
Vo],rme 1, ~mntmx 5 -
F'JSBB LETT~_RS
!.0
..
O~ober 1968
1.0
....
.-
35olm, ~
-
1 31D m p
.
/
~
~-""
"'.
i
I
!
~357mlJ
t
",.
~
lI "
I;
I t
~
--
.=
l
",~
,IF/ ~
,
t
; ',
r--,
z ~:~ 0 . 5
.m~ 0.5
-., /; ',
t
"-,.
"
% 0.1
0.1 270
200
290 300 WAVELE NGTH
350
270
400'
" 1 280
2 ~ 0 300 WAVELENGTH
350
40C
Fig. 3 . - - - - - B ] 2 a ~fter p ~ e througl~ a cheating resin, dissolved i n H 2 0 - -- . . . . . . . . . cystcamin= B12 a ( ~ th~augh
Fig= 2. ------B12a after passage through a chelating ze..s~, dissolved in H20. -- . . . . . . . cystezmine-Bl2a ( p z . . ~ through a chelating xesin) complex, 20 rain aftez the addition o f cy.'t~anine.
a chelating resin) compl¢, 20 rain z_qm the addition of'.he cystearr~ne. B 12;" .formation, a~1er additior, o f pP 8-9 buffer ~ the pjr~ence of C~++ 3 X lO -7 M. I ~- after 2 mi~; 2 = after 4 rain; 3 = after 6 rain; 4 = a~-ter 8 rain; 5 = after 10
- splitting of the cysteamine-B12a complex, aft¢~ ri.~ng the pH to 8=9; 1 = after 2 ~in; 2 = aftez 4 rain; 3 = afro': ] 0 rnin; 4 = after 20 rain; 5 = after 30 rain; 6 = after 60 rain.
nfin; 6 = afte~ 20 rain; 7 = z ~ e r 30 rain; 8 = m-to; 45 mir.; 9 =
after 60 ram.
s u m p t ~ e evidence for the necessity o f a metal ion im-
purity as an intermediate for the reduction o f E~2a, we
have eliminated eventual metals by passage o f ~3e
the reducticn o f B l 2 ~ b y cysteamJne has t o b e related with the removal o f a metal ion, the e x p e m a e n t zep o r t e d in fig. 2 has been repeated h-t the presence o f C'u++ i o n s As seen in fig. 3, the addition o f a trace o f Cu " ~ ions reintrofluce~ the reduction o f B]2 a b y c y s t e a m i n e . F e -~-~, Co - ~ , iNl-++ and Zn ++ have been assayed in the p]ace o f Cu ++, w i t h o u t ~LtCCe~. The ~ e c e s s i ~ for a metal ion has been observed also when
B ] 2 a solution through a chelating resin ( D o w e x A I in t h e Ha + form). Using the solution o f B 1 2 a passed through the chelating resin, t h e complex thiol-B12 a at p H 4.2 is again produced u p o n t h e addition o f c y ~ t e nmine (fig. 2); however, w h e n the p H o f Lhe solution is raised t o 8 . 9 no reduction occurs. In t h e place o f t h e c y s t e i n e o r m e r c a p t o e t h a n o l w e r e used i n t h e p l a c e curve o f Bl2r t h e curve is changrd ~ o w l y t o tlmt o f o f ¢ysteamme. BI2 a a t p H above 7. This observa~on ~ d l o a t e s t h a t the ]n t~e l ~ h t o f the ze.s~t~ reported above it apl~axs ~ t i o n in-the absence o f a rectal i o n produces- ' t h a t t h e reduction-of BI2 a b y th~ols is indirect..'ih =. t h e cleavage o f - t h e thi'oi-B12 a c o m v ; e ~ i n t h e p l a c e o f " presence o f a metal-ion is nece&~ry and a po~_~le exreducing BI2 a t o B l 2 r - I d e n t i c a l res~dts have been 0]3- * planation is t h a t t h e ffdol red~3ces the metal which in tahied w h e n Bt2a-fi'ee t r o m contamina~g.met~_i~ was • its turn ~ednces t h e c o b ~ l ~ m ~ n . T h a t t h i s is a reason.p r e p p e d using p.~per, elect~oph0resis in, 0.5. m acetic. _ -
"
a c i d. ] a 3 0 r d e r - t O e s t a b ~
"whether the
~uppress3on
of
-
27~
- ~olmme 1, ~umber 5
FEBS LE'rrERS
thai, u n d e r t h e conditions o f t h e l~esent expe~i~e~ts, C u + i n t h e form o f C a ~ disso]ved in 0.25 M KCTI actual]y reduces B12 a 1o B t 2 r in ~he ~ s e n c e o f a thiol at a Although t h e addition ©tz ~c.e o £ C ~ "H" ions m a y 1eplace t h e m e t ~ _f~moved b y the passage o f t h e B t 2 a solution through t h e r.~helating xes~n, we d o n o t so f~r ~:rave a n y evidence t o establish which metal is a c h ~ i l y present as an i m p m i t y in BI2 a cata]ysing t h e Teduction o f ~he coba'Jzmin b y thioIs.
274
O c ~ 0 ~ t96E
Refe~rences
~I] ~ ~ d R.Mu~ie, ] o ~ S~te L s ~ 26 (1952) s s s . [2] :].W_Ecabnoff,Bioahem. B i o p h ~ R~'~ Co~amun. 16 { 1 9 ~ ) 484. |3] M.Adler, T~edwi~k aha T.S~ozna~ki, J, Am. Chem. Soc. ZB (1966) 5 0 1 8 . " 14] Ji.Yo01, Bio~mm. J. 88 (1963) 295:, [~] E.A.Kackza, D.E.Wolf, F.~Kuehl Jz. and ICFolkers, Science 112 (1950) 354.