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A light-stimulated production of an oxidant by pyridoxal phosphate, Mn ++ and amino acids
AR(‘HIvES
A
OF
HI(I(‘HEMISTRY
ANI)
HIOPlIYSI(!S
Light-Stimulated Phosphate,
93,
:%)fi-:j11
Production
of
Mn*
and
(1!)61)
an
Oxidant
Amino
by
Pyridoxal
Acids
The combim~l ion of pyridoxd phosphatr!, MI]-+, :llld v;rrious :mino wids h.s Imm found 1.0 protloc*c an oxidant non-cnzymidly at. ncq~lrd pH. Th llw:~s~~ro of oxithtnt prothdon was III~I~~~ using I- :w th(l rcduct:mt.. The osid:~tion of I- was &ndntrtl by light. So other ndd ion t,c!sled cold rnpl:u~c Mn”. .1 wvnll-tldinctl optimum of adivity was oht;Gnetl in phosphate huffer at about pH 6.6. The oxid:mt w~fls npparcntly not hytlrog!c~n I)croxidc rrincc (*:it:tl:1s:(~ did not inhil)it. 1. oxithltion. INTRODUCTION
mixtures containing 1)yritloxal pl~oalhatr, hln t- +v anti wrtain amino acicls. This pap(‘t is a rc~l)ort, of some of tlw rliaraetcristics of t,liis reaction.
Many non-cnzymic reactions wliicli arc analogous t.0 known cnzymic transformations have heen shown to occur in t,hc prwrnce of pyridoxal or pyridoxal pllosplh~, amino acids, and various metal salts (1). An oxidatirc dcamination of pyridoxaminc and of many u-amino acids by pyridoxal and various metal ions has hen demonstrat,ed 12). An aqueous solution of pyridoxal pl~ospliate on cxposurr to light, will undergo a henzoin condensation of which tlw product is rapidly oxidized by nt,moslheric oxygen (3). A recent study (4) has shown that, the dinitrophcnyl dcrivatiws of amino acids, which can he vicwcd pch~ps as distant. relatives of tlic azomctliinc coiiiljounds formed by pyridoxal and amino acids, arc decomposed by light to yield S-alkyldinitroanilines. and volatile co,, acidic products. During the course of an invcstigat,ion of tlw sulfur amino acids as potential precursors of some of tlic clinractcri& flavors and odors of vegetahlcs, the non-enzymir oxidation of I- was olwwwd at room tempersturc and neutral pH in reaction
M:ITISIII.~LS Th Pllllh’P
st:mtl:u.tl plloPphnll?
AND
METHODS
rc;~d ion mixt.urc hfh
pIi
7.0,
c~on~ktctl 0.5
plnolo
of
166
MllCl,
,
1 ~inol~~ pyritloxd 5’-phosph:dc, 10 pmolrs ICI, 2 pmoles ;imino :Id, ;mtl tlistillctl w:il.nr t.0 :L fin;ll VOII~N: of 2 1111. Thr mixf.urc \V:IS in~1Ib:~t.d in :I twl tribe al rooin tc~ii~pcri~iu~~c with conslant shdiing undrr :I, fllmrrscrnt light, whidi prod~~c~~l :in intensity of :ilmrlt 30 ft.-~:llldlCS at the nrlrf:tc7, of l.hfx rfwr1 ion mixt.rlrr~. ;\ftc>r 90 min., 0.5 ml. of :t 0.001 :I[ iiminoni~im n~ol?:l~~l;~l~~ solution in 3 :\r H,SO I ,v:,r; :ltltlcd to 0:ldl hlh! followvod by 0.5 ml. of 6% st:irch sohlt.ion and 3 ml. of tlistillrtl wvator. After 10 min., (ho :ihsorption wiis 1nc:1srI1wl al: 600 nip in :L ~l~r~trophotomctcr. This i;; ;l slightly motlifid vcGon of t,he mclhotl for I:;- of Sitvagr (5). -4 stnritlnrti Curve for I:,- \V:is linc7ll from 26 to 246 m~molcs. Rclos 20 mpmolcs, 1 hcrc: WilP smnc dcviiilion, but lhe c-leviation wy:ilj (eonrisifwl :ind viihl(~s below 20 mpmoles could bc rc~:icl wit II r(*;ls
’ A IAboratary of tho Wcslcrn TTtilix:lt.ion Rcsearch and Development Division, Bgricultnral Research Service, U. S. Department of Agridture. 306
pmoles
Pyridoxal
Phosmhate
308
ZELIS
MA
Tltc~ 1)ossit)ility that I2 could react wit11 cotttp0nmt.s of tlta wnction tuixturc wits r’xplot~ctl. At1 nc~ucoll 2: iodine solution was mttlc ttl) wntaining 50 ntptnolcs I,,hnl. :is :iss:iycd c’olorititCt.~ic~~ll~in tltc prcwncc of TV. ‘I’llc wsults of twting to the rcsctivity of tltr various coiiiponcnts of tltc usu:~l w:tction tuixturct with I2 :trc shown in Table III. In vwry ciwc ivltwc 1)yridoxal phoslhtc wls lmwnt~ Init. Mu++ or the amino :wid :ilw:cnt, tlw t~tttouiit of T, rrm;tining after the usual incuhit~ioti tiuic was ncgligilh. Only wlwt~c all tlw ~otuponents wew Inwont n-as my ;tpprcviaMc :ttitount of tlw 1, a~hlcd itiiti:rllv rrt:lincd. Again, if tyrosine wis th Ano avid, :i rapid (lccolor:~tiori of tlw l)lue stnrrlt-iotlinc conqhx ovwtwtl. Thit tltis w:ib not, tliw to tyrosine as swlt wiw ~liowii hy aclding tytwinc to :I coui~~l~tc~ waction itiisturr ctscq)t for amino witI to wltivlt I:{- I~ntl lwii :itlded and thrn tlw
ul.-~tfdiioiiitie lx-Ncthiottitte osidr o~-iUd hiottirte
II lJl.-l’rypt 3!) ! L-Glttl.:ttttic I 107 I.-GIu1:ttttittc
stdfsu-
I3 X0
oph:m
witl
2.5
font
JIetlt?-ltttcthictttitt(~ foniwtt rhloridc L-Cywtcittc ur,-Horttoc,~steille AS-JIrl,li~l-I.-~~nt.eitt(: S-IIct hyl-I.-cyst.c!iitr sitlfoxidt! ul,-Swi ttf: I,r.-IIottioscritio UL-Thrwtiitic t)t.-l’hctt?.ltLI:tttitte nI,-‘l’\-rosi tic t~1~-I~ih~dr~~s~Jtltc~it~~dattiite I.-llistillitto
su-
8
I,I,-wNH~N-
X
tj7G: :tcicl 13J s-SH4utgric 0 :tcid 3 I I.-Aslxtri,ic x&l :I i I.-th/,ccl-8-blcl:hyl :ispart ic: acid I60 I.-t’rtrlitte ut,-Al:triitte 28 4 Ij-A~:ttiiite 11 t)t.-Lctwitic 35” (;I.wi tic 45 ur.-\;:tlitic 10
2 I1 ‘, 3 36 2 8 !I 4
r)t.-i,.vsittr
(i
s;tal~call
:ts 1lsll:ll
:1ntl
t1w
color
IdlIe
fol~lllrll.
So fwling owurrctl under tltrw conditions. after
I2 Blw st:treh color red :tl (310 tttj.4 itttttiedi:ttd~ rt:irch adtlitiott itistc:ttl of IO tttiit. :iflcr.
sinr. In tltc! t~x~t~~l)l~in T:tl)lc II, ttfkt’ 10 min. tlto tyrosinc twtding gtlw only 5 uiptttolw of I:$-. Tlic f:itling of tlw color did not occur witlt my 0th miino acid.
Tlie non-onzyniic tttoclrl Iwctions twtw-crn p~ritlosiil and :itttino :icicl* such an t.l.:tii~t,liiin:ltioa arc ratalyzctl by 3 vsrkty of uichl ions, including (;a+++, Cu++, A\li-Lt-s Fc+T! Fp+++, z11++, In++, Si- -‘-, ;ind Co+ f. In gwwr:ll, tltc ardor of cff~~tirmcss wts its listed frotn most. &cc.tivv to lcttst (1 I. Tltc oxidation of I- as a function of the nictal ion prcscnt wis (lx2tniincd. Srrinr~ and tttc4l~ioninc sulfoxidc w-erc utilized iis tlict mtino ;tcids. In citlwt r;iw, no iiictal ion could rcplaee Mn+ +. Fc++‘, Fo++, Cut-i. %n++. ‘A]++’ (:o-!--.,
1\Ig++,
‘CR++,
Sn++,
arl(l
xi++
WYT tcs:tcd. Only Co++ had any effect, ant1 only 2% :is &e&w 85 Ah++.
it. w:w
%YWIA’lWJ~
I
I,l(;lI-f’
OP
I:,-
FORMATIOS
Th titttc :~llowcd for t.ltr wwtion w:ls usu:~ily 90 ttlin. In T:thlc I, it, ran he seen that iii t.lw ww of ntctltioninc sulfom, unlilw :il:initic, scrinc, or nwthionitw wlfoxid~~.
Coinpletc reaction tttixt.tirc tttntle up :tn ticswrilwd in ~Urthodu 8wtion cwept 50 tnfint0lc.r I:! atidctl inslcad of 10 pttioles ICI. At end of incul)sCon t.itne ICI was :tdtled :md 1he starch-l:{ color tlcvelopcd :is tlescril)ed previoitsly.
Reaction minturr ----_ Cotnplctn, 3litttts .\/Iittus Mitttiri Cottiplt:tr. ~!ontplc!.r,
10
ntcthionittc
mjdmotes Ia- formed -. ..-.. sttlfotte
2!)
MII-”
tnet~hiortiti~ pyridosd alanine t yrosine
4
sttlfottc phosphate
2; 26
28”
n >Iexatrod ;tt ottce afkr starch :tddii.ion. min. t.he v:tIue had decressed to 3.
Aft.at
Gl50, z rt
E
100;
50--
310
MAZEIJS
the duplicates was evacuated on a water acid is absent indicates that a reaction :tq)irator for 10 min. The rraction was occurs when all the components are present startrcl by tipping in the pyridosal phos- which makes t,lle pyridoxal plloephat~eunphate in t,lic aerobic tube and the cvacu- available to the iodine. Also! the fact t,hat atccl t.ubc. After 90 min. under t.hi: light with at higher pi-l in l)hosphatc bufler the addiconstant shaking, the molybdate-H,SO, tion of mow 3~111.~ + does not readily form was tipped in, the wssrls olwnccl, and the: a pwcipitatc in the>1uwoncc of amino acid starch and distilled n-at.cr uddcd as usual. antI 1)yritlos:il pliosphatc iniplic~s that the Ko I- oxidation owurrcd uncicr anaerobic Mn t-+ in rcfitaincil in solution 1)~ c1~c:lat.c condit.ions. That. this was not, a rate cffcct formation. n-as shown by increasing tlit t,imc of incuThe rcsulta in Table II show that timall bation and finding no I,- formation in an rh:tngw in the amino acid structure cause large variations in the oxidant-forming caanaerobic environment in any case. pa&y. In tlic nict,hioninc scriw, it, is swii t,hat, as the sulfur becomes mow highly The results obt,aincd when pyridoxal is oxidized, the ability to generate more oxi*ubstit~utcd for pyridoxal pliosphatc are dant is incrcnsed. This could bc cxl)luinvd strong evidence that the carbonyl function on the grounds that the rcduccd sulfur of is ncwssary for t,hc oxidat.ion to t.ake place. nwtjliioninc* and the relatively riducwl WIAt, neutral pH, the hcwiacet.al form of fur of the sulfoxide are competing with tlw pyridoxal is predominant. (1) . Thr phospatc iotlidc for thcl oxidant produced. Thr eulsubstitution in pyridox:ll phospliatc does font? having alwady a completely oxiilizc~tl not allow the forim~tion of the internal sulfur, tlow not, cwtcr this comprtitiou ant1 Iiemiatcctal to occur. If the carbonyl func- thcxiotli&* is the role rAwtant.. Thv cfftvt of tJon is prcscnt., then the: formation of tile ii nii4.liyl~ni~ group is shown b;v the corn:~zomcthinc intc3wiediat.c with any amino parison of hoinoc~ystcinc which is innctivc arid present should occur, and it would md cystiGni~ which is very activ-c. Jlctliylswni logical that this intcrnircliatc~ is the at.ing hoiiiocy&c~inc~ t,o mcthioninc and its actual compound t,hat is somcliow inrolwd cli~riwtiws iniwasw the oxidnnt.-1,rocluririg in oxidant production. The sprcifirity for ability ; t,lic rcwrsc is seen in the cyat~c4nv .I Nn ‘--I- is an intcrceting fcntuw. In all the t.0 S-iiictlivlc~.~tririe scrics. Svrinc :in(l other non-enzymic nwtal-ion pyridoxahoiiioscrinc~’arc similar to t,licir tliio :uwamino acid reactions, there was a gradation logs. An intcrcsting study of struct.urc as of activity with different metal ions from related to oxidant formation is wiw in tlic wry active to inactivr. The prcscnt (YW:C’ glut.:uuic* acid group. Glutamic acid i$ vwy shows no art.ivit,y wit.h any metal ion saw artivc. Tf the y-carboxy is rw~ovctl t.llc hIn+ +. The small amount of activity found nrtivity is lost; if the a-carbosyl group is with (lo++ could likrly be c~xplaincvl by a wiiiovrd, tlw activity is also lost; if tlic small Ma + -1.contamination in tlic rcagiwt, y-r:1rl,0sg1 is aiuiilat~cd, t,lic activity i$ sincr no purificnt,ionl; were mnclc of the ~tu~rkrrlly tlrcw:~r;ctl. The aronnltir amino mrtal sa1t.suscvl. The quwtion as to wllc$Jle~ a&l wriw also ~1~0~stliil incrcwing foriiiat.lie Mnl+, pyridoxal phosphate, and amino tion of OXiflilIlt as the aromatic ring Iwonic~s a&l form ii pyridoxylitliw-metal rhclatc more oxitlizctl. There is an incrcasc in osia:: has been dcmonstratcd for 01-&t- (6) , tlant, Iwodurtion from 1)hrnylalnninc t.o Xl -1-1-+I 6 I am1 Xi-t .+ i7 I with 1)yridoxal i~ili~~tlroxypl~c~~iyl~~laiiinc. The vanishing ant1 alaninch was not. di~uionatratccl un- color with tyrosinr is difficult to cvqGn. equivocally. Certain facts provide sonic As 1)ointwl out, prwiously it is not diii~ to reason:Nr prceuinptirc widenw for tliil tliv tyrosine itsclf. Soiric~ iutwiiwcliatc~ formation of such a co~nplm. The fact that fornwd during tlw reaction is reqwwibIc I, will watt with thih roiuponcnts of t,lii? for thr instabiltiy of the color. This intchrnwtliatr i?;iiniqiw for tyroainr as the aniino riwtion iuixtuw in the prcwncr of pyridoxal phosphat~i~ only if Mn+ + or the amino acid.
liillitiiig in tlic over-all rcwction :ind the fotmltiun of which would lx ligh t-etinlu1:itcvl. (hwc~ the concmtration of intwulc~cli:itv wis high rnougli, thei-c would lw no r*ffvrt of light, on tlw over-all wwtion. The wtunl oxidant is apparently not liydrogcn ~woxitlc ainct~ ratalaac had no oticct. Tlw :irsunll)tion has been inadr that t.lic osirlniit formr~tl is the sanw rcgrtrdlcss of tlw nmino a&l Iwwnt, or whet,lwr in thr light, or dnrk. This :wsuiuption may not, lx justifirtl.
1. 2. 3.
4. 5 6.
7. 8.
9.
A
OF
HI(I(‘HEMISTRY
ANI)
HIOPlIYSI(!S
Light-Stimulated Phosphate,
93,
:%)fi-:j11
Production
of
Mn*
and
(1!)61)
an
Oxidant
Amino
by
Pyridoxal
Acids
The combim~l ion of pyridoxd phosphatr!, MI]-+, :llld v;rrious :mino wids h.s Imm found 1.0 protloc*c an oxidant non-cnzymidly at. ncq~lrd pH. Th llw:~s~~ro of oxithtnt prothdon was III~I~~~ using I- :w th(l rcduct:mt.. The osid:~tion of I- was &ndntrtl by light. So other ndd ion t,c!sled cold rnpl:u~c Mn”. .1 wvnll-tldinctl optimum of adivity was oht;Gnetl in phosphate huffer at about pH 6.6. The oxid:mt w~fls npparcntly not hytlrog!c~n I)croxidc rrincc (*:it:tl:1s:(~ did not inhil)it. 1. oxithltion. INTRODUCTION
mixtures containing 1)yritloxal pl~oalhatr, hln t- +v anti wrtain amino acicls. This pap(‘t is a rc~l)ort, of some of tlw rliaraetcristics of t,liis reaction.
Many non-cnzymic reactions wliicli arc analogous t.0 known cnzymic transformations have heen shown to occur in t,hc prwrnce of pyridoxal or pyridoxal pllosplh~, amino acids, and various metal salts (1). An oxidatirc dcamination of pyridoxaminc and of many u-amino acids by pyridoxal and various metal ions has hen demonstrat,ed 12). An aqueous solution of pyridoxal pl~ospliate on cxposurr to light, will undergo a henzoin condensation of which tlw product is rapidly oxidized by nt,moslheric oxygen (3). A recent study (4) has shown that, the dinitrophcnyl dcrivatiws of amino acids, which can he vicwcd pch~ps as distant. relatives of tlic azomctliinc coiiiljounds formed by pyridoxal and amino acids, arc decomposed by light to yield S-alkyldinitroanilines. and volatile co,, acidic products. During the course of an invcstigat,ion of tlw sulfur amino acids as potential precursors of some of tlic clinractcri& flavors and odors of vegetahlcs, the non-enzymir oxidation of I- was olwwwd at room tempersturc and neutral pH in reaction
M:ITISIII.~LS Th Pllllh’P
st:mtl:u.tl plloPphnll?
AND
METHODS
rc;~d ion mixt.urc hfh
pIi
7.0,
c~on~ktctl 0.5
plnolo
of
166
MllCl,
,
1 ~inol~~ pyritloxd 5’-phosph:dc, 10 pmolrs ICI, 2 pmoles ;imino :Id, ;mtl tlistillctl w:il.nr t.0 :L fin;ll VOII~N: of 2 1111. Thr mixf.urc \V:IS in~1Ib:~t.d in :I twl tribe al rooin tc~ii~pcri~iu~~c with conslant shdiing undrr :I, fllmrrscrnt light, whidi prod~~c~~l :in intensity of :ilmrlt 30 ft.-~:llldlCS at the nrlrf:tc7, of l.hfx rfwr1 ion mixt.rlrr~. ;\ftc>r 90 min., 0.5 ml. of :t 0.001 :I[ iiminoni~im n~ol?:l~~l;~l~~ solution in 3 :\r H,SO I ,v:,r; :ltltlcd to 0:ldl hlh! followvod by 0.5 ml. of 6% st:irch sohlt.ion and 3 ml. of tlistillrtl wvator. After 10 min., (ho :ihsorption wiis 1nc:1srI1wl al: 600 nip in :L ~l~r~trophotomctcr. This i;; ;l slightly motlifid vcGon of t,he mclhotl for I:;- of Sitvagr (5). -4 stnritlnrti Curve for I:,- \V:is linc7ll from 26 to 246 m~molcs. Rclos 20 mpmolcs, 1 hcrc: WilP smnc dcviiilion, but lhe c-leviation wy:ilj (eonrisifwl :ind viihl(~s below 20 mpmoles could bc rc~:icl wit II r(*;ls
’ A IAboratary of tho Wcslcrn TTtilix:lt.ion Rcsearch and Development Division, Bgricultnral Research Service, U. S. Department of Agridture. 306
pmoles
Pyridoxal
Phosmhate
308
ZELIS
MA
Tltc~ 1)ossit)ility that I2 could react wit11 cotttp0nmt.s of tlta wnction tuixturc wits r’xplot~ctl. At1 nc~ucoll 2: iodine solution was mttlc ttl) wntaining 50 ntptnolcs I,,hnl. :is :iss:iycd c’olorititCt.~ic~~ll~in tltc prcwncc of TV. ‘I’llc wsults of twting to the rcsctivity of tltr various coiiiponcnts of tltc usu:~l w:tction tuixturct with I2 :trc shown in Table III. In vwry ciwc ivltwc 1)yridoxal phoslhtc wls lmwnt~ Init. Mu++ or the amino :wid :ilw:cnt, tlw t~tttouiit of T, rrm;tining after the usual incuhit~ioti tiuic was ncgligilh. Only wlwt~c all tlw ~otuponents wew Inwont n-as my ;tpprcviaMc :ttitount of tlw 1, a~hlcd itiiti:rllv rrt:lincd. Again, if tyrosine wis th Ano avid, :i rapid (lccolor:~tiori of tlw l)lue stnrrlt-iotlinc conqhx ovwtwtl. Thit tltis w:ib not, tliw to tyrosine as swlt wiw ~liowii hy aclding tytwinc to :I coui~~l~tc~ waction itiisturr ctscq)t for amino witI to wltivlt I:{- I~ntl lwii :itlded and thrn tlw
ul.-~tfdiioiiitie lx-Ncthiottitte osidr o~-iUd hiottirte
II lJl.-l’rypt 3!) ! L-Glttl.:ttttic I 107 I.-GIu1:ttttittc
stdfsu-
I3 X0
oph:m
witl
2.5
font
JIetlt?-ltttcthictttitt(~ foniwtt rhloridc L-Cywtcittc ur,-Horttoc,~steille AS-JIrl,li~l-I.-~~nt.eitt(: S-IIct hyl-I.-cyst.c!iitr sitlfoxidt! ul,-Swi ttf: I,r.-IIottioscritio UL-Thrwtiitic t)t.-l’hctt?.ltLI:tttitte nI,-‘l’\-rosi tic t~1~-I~ih~dr~~s~Jtltc~it~~dattiite I.-llistillitto
su-
8
I,I,-wNH~N-
X
tj7G: :tcicl 13J s-SH4utgric 0 :tcid 3 I I.-Aslxtri,ic x&l :I i I.-th/,ccl-8-blcl:hyl :ispart ic: acid I60 I.-t’rtrlitte ut,-Al:triitte 28 4 Ij-A~:ttiiite 11 t)t.-Lctwitic 35” (;I.wi tic 45 ur.-\;:tlitic 10
2 I1 ‘, 3 36 2 8 !I 4
r)t.-i,.vsittr
(i
s;tal~call
:ts 1lsll:ll
:1ntl
t1w
color
IdlIe
fol~lllrll.
So fwling owurrctl under tltrw conditions. after
I2 Blw st:treh color red :tl (310 tttj.4 itttttiedi:ttd~ rt:irch adtlitiott itistc:ttl of IO tttiit. :iflcr.
sinr. In tltc! t~x~t~~l)l~in T:tl)lc II, ttfkt’ 10 min. tlto tyrosinc twtding gtlw only 5 uiptttolw of I:$-. Tlic f:itling of tlw color did not occur witlt my 0th miino acid.
Tlie non-onzyniic tttoclrl Iwctions twtw-crn p~ritlosiil and :itttino :icicl* such an t.l.:tii~t,liiin:ltioa arc ratalyzctl by 3 vsrkty of uichl ions, including (;a+++, Cu++, A\li-Lt-s Fc+T! Fp+++, z11++, In++, Si- -‘-, ;ind Co+ f. In gwwr:ll, tltc ardor of cff~~tirmcss wts its listed frotn most. &cc.tivv to lcttst (1 I. Tltc oxidation of I- as a function of the nictal ion prcscnt wis (lx2tniincd. Srrinr~ and tttc4l~ioninc sulfoxidc w-erc utilized iis tlict mtino ;tcids. In citlwt r;iw, no iiictal ion could rcplaee Mn+ +. Fc++‘, Fo++, Cut-i. %n++. ‘A]++’ (:o-!--.,
1\Ig++,
‘CR++,
Sn++,
arl(l
xi++
WYT tcs:tcd. Only Co++ had any effect, ant1 only 2% :is &e&w 85 Ah++.
it. w:w
%YWIA’lWJ~
I
I,l(;lI-f’
OP
I:,-
FORMATIOS
Th titttc :~llowcd for t.ltr wwtion w:ls usu:~ily 90 ttlin. In T:thlc I, it, ran he seen that iii t.lw ww of ntctltioninc sulfom, unlilw :il:initic, scrinc, or nwthionitw wlfoxid~~.
Coinpletc reaction tttixt.tirc tttntle up :tn ticswrilwd in ~Urthodu 8wtion cwept 50 tnfint0lc.r I:! atidctl inslcad of 10 pttioles ICI. At end of incul)sCon t.itne ICI was :tdtled :md 1he starch-l:{ color tlcvelopcd :is tlescril)ed previoitsly.
Reaction minturr ----_ Cotnplctn, 3litttts .\/Iittus Mitttiri Cottiplt:tr. ~!ontplc!.r,
10
ntcthionittc
mjdmotes Ia- formed -. ..-.. sttlfotte
2!)
MII-”
tnet~hiortiti~ pyridosd alanine t yrosine
4
sttlfottc phosphate
2; 26
28”
n >Iexatrod ;tt ottce afkr starch :tddii.ion. min. t.he v:tIue had decressed to 3.
Aft.at
Gl50, z rt
E
100;
50--
310
MAZEIJS
the duplicates was evacuated on a water acid is absent indicates that a reaction :tq)irator for 10 min. The rraction was occurs when all the components are present startrcl by tipping in the pyridosal phos- which makes t,lle pyridoxal plloephat~eunphate in t,lic aerobic tube and the cvacu- available to the iodine. Also! the fact t,hat atccl t.ubc. After 90 min. under t.hi: light with at higher pi-l in l)hosphatc bufler the addiconstant shaking, the molybdate-H,SO, tion of mow 3~111.~ + does not readily form was tipped in, the wssrls olwnccl, and the: a pwcipitatc in the>1uwoncc of amino acid starch and distilled n-at.cr uddcd as usual. antI 1)yritlos:il pliosphatc iniplic~s that the Ko I- oxidation owurrcd uncicr anaerobic Mn t-+ in rcfitaincil in solution 1)~ c1~c:lat.c condit.ions. That. this was not, a rate cffcct formation. n-as shown by increasing tlit t,imc of incuThe rcsulta in Table II show that timall bation and finding no I,- formation in an rh:tngw in the amino acid structure cause large variations in the oxidant-forming caanaerobic environment in any case. pa&y. In tlic nict,hioninc scriw, it, is swii t,hat, as the sulfur becomes mow highly The results obt,aincd when pyridoxal is oxidized, the ability to generate more oxi*ubstit~utcd for pyridoxal pliosphatc are dant is incrcnsed. This could bc cxl)luinvd strong evidence that the carbonyl function on the grounds that the rcduccd sulfur of is ncwssary for t,hc oxidat.ion to t.ake place. nwtjliioninc* and the relatively riducwl WIAt, neutral pH, the hcwiacet.al form of fur of the sulfoxide are competing with tlw pyridoxal is predominant. (1) . Thr phospatc iotlidc for thcl oxidant produced. Thr eulsubstitution in pyridox:ll phospliatc does font? having alwady a completely oxiilizc~tl not allow the forim~tion of the internal sulfur, tlow not, cwtcr this comprtitiou ant1 Iiemiatcctal to occur. If the carbonyl func- thcxiotli&* is the role rAwtant.. Thv cfftvt of tJon is prcscnt., then the: formation of tile ii nii4.liyl~ni~ group is shown b;v the corn:~zomcthinc intc3wiediat.c with any amino parison of hoinoc~ystcinc which is innctivc arid present should occur, and it would md cystiGni~ which is very activ-c. Jlctliylswni logical that this intcrnircliatc~ is the at.ing hoiiiocy&c~inc~ t,o mcthioninc and its actual compound t,hat is somcliow inrolwd cli~riwtiws iniwasw the oxidnnt.-1,rocluririg in oxidant production. The sprcifirity for ability ; t,lic rcwrsc is seen in the cyat~c4nv .I Nn ‘--I- is an intcrceting fcntuw. In all the t.0 S-iiictlivlc~.~tririe scrics. Svrinc :in(l other non-enzymic nwtal-ion pyridoxahoiiioscrinc~’arc similar to t,licir tliio :uwamino acid reactions, there was a gradation logs. An intcrcsting study of struct.urc as of activity with different metal ions from related to oxidant formation is wiw in tlic wry active to inactivr. The prcscnt (YW:C’ glut.:uuic* acid group. Glutamic acid i$ vwy shows no art.ivit,y wit.h any metal ion saw artivc. Tf the y-carboxy is rw~ovctl t.llc hIn+ +. The small amount of activity found nrtivity is lost; if the a-carbosyl group is with (lo++ could likrly be c~xplaincvl by a wiiiovrd, tlw activity is also lost; if tlic small Ma + -1.contamination in tlic rcagiwt, y-r:1rl,0sg1 is aiuiilat~cd, t,lic activity i$ sincr no purificnt,ionl; were mnclc of the ~tu~rkrrlly tlrcw:~r;ctl. The aronnltir amino mrtal sa1t.suscvl. The quwtion as to wllc$Jle~ a&l wriw also ~1~0~stliil incrcwing foriiiat.lie Mnl+, pyridoxal phosphate, and amino tion of OXiflilIlt as the aromatic ring Iwonic~s a&l form ii pyridoxylitliw-metal rhclatc more oxitlizctl. There is an incrcasc in osia:: has been dcmonstratcd for 01-&t- (6) , tlant, Iwodurtion from 1)hrnylalnninc t.o Xl -1-1-+I 6 I am1 Xi-t .+ i7 I with 1)yridoxal i~ili~~tlroxypl~c~~iyl~~laiiinc. The vanishing ant1 alaninch was not. di~uionatratccl un- color with tyrosinr is difficult to cvqGn. equivocally. Certain facts provide sonic As 1)ointwl out, prwiously it is not diii~ to reason:Nr prceuinptirc widenw for tliil tliv tyrosine itsclf. Soiric~ iutwiiwcliatc~ formation of such a co~nplm. The fact that fornwd during tlw reaction is reqwwibIc I, will watt with thih roiuponcnts of t,lii? for thr instabiltiy of the color. This intchrnwtliatr i?;iiniqiw for tyroainr as the aniino riwtion iuixtuw in the prcwncr of pyridoxal phosphat~i~ only if Mn+ + or the amino acid.
liillitiiig in tlic over-all rcwction :ind the fotmltiun of which would lx ligh t-etinlu1:itcvl. (hwc~ the concmtration of intwulc~cli:itv wis high rnougli, thei-c would lw no r*ffvrt of light, on tlw over-all wwtion. The wtunl oxidant is apparently not liydrogcn ~woxitlc ainct~ ratalaac had no oticct. Tlw :irsunll)tion has been inadr that t.lic osirlniit formr~tl is the sanw rcgrtrdlcss of tlw nmino a&l Iwwnt, or whet,lwr in thr light, or dnrk. This :wsuiuption may not, lx justifirtl.
1. 2. 3.
4. 5 6.
7. 8.
9.