- Email: [email protected]
N-terminus of α-crystallin
Exl)tl lgye Res. (1968), 7, 291-300
N-Terminus of m-Crystallin FI. ,J. ]Iot.:~m.:~s, 3. O. G. SCHOV;X,~AKmtS, J. J. 'l?. C,~.~r~D~r~c,, G. I. T~.:SSI~R* .~.~t) l I. BLO~++~J..:m)At,
Department of Biochemistry, University of Nijmegen, N0",te.qen , The Netherla.nds (Pape, f,om the Symposh+m.
Lc,es Proteins and Relatexl Subjects", Ghent, Belgium, June 1967)
:k p u r e N - t e r m i n a l p('pt.i(I¢: wa,,+ i~olal:(.-d f r o m b o v i n e l e n s ;¢.-crystallill ,~,l+ter prono, se d i g e s i i n n , eat.ion e x c h t t n ~ e on l ) o w e x 50~V-X2 ~uxd t w o atldibiom~l e l e c t r o l ) h o r e t i c purific~t, iott s t e p s . :['he c ! ! w i d a l i o n o f l.he sl, r u c g u r e b y m e a n s o f h i g h volg~+ge eleel~rophoresis, t h i n l a y e r e | u ' o m a u o g r ~ p h y , g a s c h r o m ' t t ( ~ g r ~ l ~ h y tutd. mtcle0,r magn.et, ic rosotl~xrtce speot, r o s e o p y r e s u l t e d in t h e t'ormul~v: z'V-acct,yl-3[el,-Asp. <)~t~; a('+,tyl grotl|) xvtis foull(! p e r a p p r < ) x i m a t c l 5' 2 1 , 0 0 0 g o f ~ - c r y s t M l i n . T h e l a t t e r v a l u e is t-~n~sistent, w i t h tht~ mt~h.cttlar w e i g h t o f ghe s n b m x i t s . 'l'ht~ p r e v i o u s f i n d i n g s t h a e g l u t . ~ m i e a c i d w a s t h e N-t
i. Introduction Since 1957 a mtml)er of laboratories rcl)orted t.lu~t glut, amic acid was t h e N - t e r m i n a l arnino aci~l of bovim, le,~s ~-erys.tallin (].~loemendal, 1957; B l o e m e n d a l and t e n Cate, 1958; ]°irfarova. ] 958; H/thncl, ]96]; v~,t .Dam a,nd ten Care, 1966). I,t r('ecld: ve:~rs the (le-q'ec of ]mrit.y of a.-cryst.,dlill p r e p a r a t i o n s has been iml,roved by lneans of elcetrophoresis, d e n s i t y g r a d i e n t centrifugation, p r e c i p i t a t i o n techniques and ,gel tilt:ration. I n t;hesc highly purified a.-crystallin :preparations o,gy one inch. of N - t e r m i n a l g l u t a m i c acid per a p l ) r o x i m a t e l y 2.]06 g of p r o t e i n could be detected (Blo,,me,ula], B e n t and Be,mdctti, 1965; Bloemendal, .I-Ioenders, Schoenmakers, Zwecrs altd Gerding, 1966). A p p a r e n t l y the larger a m o t m t s of N - t e r m i n a l g l u l a m i c acid prcviolmly observed were p r o b a b l y caused by cmlt,~n~ination, sitme practically a.II of it co~dd be removed, especially by gel filtration. ]31oemendal, ]3ont, J o n g k i n d and Wisse (1962) dcmonstrz~ted t h a t 0¢-crystallin (molecular weight approximate.]y 800,000) possessed a q u a t e r n a r y structure and t h a t it could be reversibly dissociated into sttbunits or p o l y p e p t i d e chains with a molecular weight of a b o u t 25,000. '+['hlus for cach s u b m d t the amouul5 of D N P - g l u t a m i c acid present is h a r d l y siguiticant and could be desigmtted as a trace end group. One e x p l a n a t i o n of this curious fact is t h a t tim N - t e r m i n a l anlino acid of ~.-crystallin was inaccessible to t h e reactiotx with 1-fluoro-2, 4-dirficrobenzene (:FDxNrB). The possibility t h a t t h e N t e r m i n a l a m i n o acid was blocked by a c a r b o h y d r a t e or an acetyl group was suggested by Bloemendal et al. (1965). I n a symposium, ~,Valey (1966) eommmxieated t h a t q u a l i t a t i v e analysis of pronase digests of c¢-eITstMli~l showed the presence o£ a blocked t e t r a p e p t i d e with m e t h i o n b l e as the N - t e r m i n a l amino acid. We d e t e c t e d a blocked dipeptide in digests of v.-crystallin in n u m e r o u s ex~erimelxts. I n t h e p r e s e n t paper vim composition of the dipeptide and the n a t u r e of t h e blocking g~'oup recovered in c¢-erystallin digests is reported. A p r e l i m i n a r y a c c o u n t has been given elsewhere (Iq:oenders a n d :Bloemendal, 1967). ~<1 3 e p a r t m c n t o f Organic C h e m i s t r y , U n i v e r s i t y o f -.-N~!jmcgcn, N i j m e g e n , T h e N e t h e r l a n d s . 291
•2q2
1l. J . H O.I':XDF. IIS I'.'T AL
2. ~ i a t e r i a l s and N I e t h o d s
Prej~aration. of lens extract Calf e y e s were ol)tailm~l f r e s h f r o m the s/aught.erhouse a n d the lenses were r e m o v e d i r a , h e & l a t e l y F i v e milliliters of w a t e r were a d d e d to l g ot" It.uses. T h e war, or-soluble protein.s were e x t r a c t e d i~l a cold room by g e n t l e s t i r r i n g until t:he c o r t e x was dissolved. T h e I.m-bid s o l u t i o n was c e n t r i f u g e d fur 10-15 rain at. 25,01X):.: g a t a low tmnlmrat.ure. T h e r e s u l t i n g c l e a r s o l u t i o n was l y o p h i l i z e d a n d s t o r e d a t --25°C.
Isolation o.f c,.-ctT/stalli.n T h e m e t h o d d e s c r i b e d by S p e c t o r (19114), with s o m e modiiicat.ions, was used as the lirst p u r i f i c a t i o n step. All m a n i p u l : ) t i o n s were 1)evformcd at. 0-5~'C.. One millilit.ev of 0.1 .~1 '['ris buffer (pi'[ 7-3) a n d 60 i~1 of t).173 5t zinc a c e t a t e , dissolved in the s a m e buffer were a d d e d to 5 ml of t h e lens e x t r a c t . A f t e r I5 rain, 0.9 In[ o( iec-coi¢[ e t h a n o l was a d d e d slow])" to t h e solution w i t h stirring. A f t e r centrifllg;ttion ab 25,0007.,g the SUl)ern:ttant fluid wa.¢ brought, to pI-I 6-5 w i t h x ItCL T h e i)recipi~ate was r e n m v c d by (,(.nrcifugation a n d t h e s u p e r n a t a n t fluid acidified w.t.th x HCI t,, tim is~mh:ct.ric p o i n t of a.-e,'ystalli~t (pl-[ 5-2). .Fbaal/v, tile p r e c i p i t a t e d m a t e r i a l was s u b m i t t e d to gel filt.rabi
Sy~ thes is of N -formyl-L- rneth io nyl~ L- aspa rt ic acid (a) Synthe-sis o/ N-formyl-t.-methionyl-r-aslmrtic ~tcid ,limeth?tl e,ster, 2"0 g (10 m n m l c s ) of L-aspart.ie a c i d d i m e t h v l e s t e r hy,'dvochh)ride ((]rnssnt;Lnn aml \Viins('h, 1958) were dis.~olved in 5 ml of d J ~ n e t h y l f o r m a m i d e a n d ([iluted. w i t h I0 nd ~f e t h y l acctat.e. '.!2o t h e nlixtu.re 1-4 ml (10 mmoles) of t r i e t h y l a m i n e w e r e a d d e d a~M the whit.e l)recipit:at#, was filtered off a f t e r 5 rain. To t h e filtrate 1.8 g (1() m m o l e a ) N-formyl-u-methi,:~ltine. ( W i n d u s a n d Marvel, 1931) were a d d e d imme,.t a t e l y to p r e v e n t i n t e r n a l a n d i u t r a m o l e c u l ' a ' cond e n s a t i o n s of t h e e s t e r t'uncGons du(*. to t.he I)rese'we .... )f .~ free amino, group, a n d 2"..'3 .g (20 m m o l e s ) N - h y d r o x y s u c c i n i r n i d e (Wiin.-:ch a n d J a e g c ' , 1966) were dissolved in t h e m i x t u r e ' ( t h i s s n b s t a n e e in.hibit,a r a c e m i z a t i o n a n d o f t e n i m p r o v e s the yield in pept.itle couplings w i t h dicyclohexylcarbodiimi(1,'.% \ V e y g a n d , IIofl'nmim a n d Wiinsch, 1966). T h e m i x t u r e w a s t h e n cooled bo ---22~'C a n d a solution of 2-20 g (10-7 mmoles) dicycl<,h e x v l c a r b o d J i m i d c in e t h y l a c e t a t e was a d d e d . The r e a c t i o n flask was k e p t "tl; this t e m p e r a t u r e for 15 rain a n d t h e n left ove/'night, in t.he refi'igeru~or a t 0°0. T h e n e x t d a y , precipit a t e d d i c y c l o h e x y l m ' c a was filtered off nnd, t.he residue was w a s h e d w i t h a s m a l l a m o u n t o f e t h y l ncet.ate. ~ h . flit.rate was c o n c e n t r a t e d in v a e u o a n d d.ilutetl w i t h c h l o r o f o r m . T h e s o l u t i o n was t h e n t,'ansferred to q s e p a r a t o r v f m m e l a n d w a s h e d s u c c e s s i v e l y with wat.er, s o d i u m b i c a r b o n a t e a n d citric :mid solutioJt a n d again with w a t e r ( a d d i t i o n of s o d i u m c h l o r i d e to t h e s e e x t r a c t i o n solutions will p r e v e n t th('. escape of a n y of the w a t e r - s o l u b l e product.s). T h e e]xloroform e x t r n e t wns d r i e d wit.h s o d i u r a s u l f a t e a n d e v a p o r a t e d t.o leave 9-.50 ~ (78~/o) or a colorless oil. T h e m a t e r i a l was used as s u c h ia t h e nexl, step. (b) Hydrolysis of the protected dipeptide ester. K y d r o l y s i s was p e r f o r m e d a f t e r dissolving of t h e m a t e r i a l in w a t e r a t p H 12 b y a d d i t i o n of 4 x s o d i u m hydro.xide. T h e s o l u t i o n was k e p t a t t k i s pt-I v a h t e for 10 rain. A f t e r r e m o v a l o f a small a m o u n t of insoluble m a t e r i a l it w a s filtered t h r o u g h a c o l u m n of s t r o n g l y acidic ion e x c h d u g e r (Amberlite I1¢-I20). T h e c o l u n m w a s w a s h e d w i t h w a t e r u n t i l the initially acidic effluent ( p H 3 - 4 ) r e a c h e d n e u t r a lity. T h e effluent w a s lyopKilizecl to leave a w h i t e a n d britt.le f o a m (1-95 g -~ 85~/o). T h i s material k e p t a t r o o m t e m p e r a t u r e t,ransformed after s o m e d a y s to a hard glassy mass. T h e acid d i s s o l v e s easily in wat.er a n d in alcohols, b u t is insoluble in n o n - p o l a r solvent, s. T h e p r o d u c t .was s u b j e c t e d to t h i n l a v e r c h r o m a t o g r a p h y on silica gel using the s y s t e m e t h y l a c e t a t e + p y r i d i n e - l - , a c e t i c a c i d + w a t e r ( 6 2 . - F 2 1 + 6 + 1 1 , b y v o l m n e ) . T h e m a i n spot,
N-'I'EI[MINU'S
OF
Cz. C R Y S : I ' A 1 .
LIN
293
eo~npri.sing abmai . q 0 ~ (d" t h e n m t e r i a l , al)p(,ar(.d a t l@ 0.32, mirror s p o t s m i g r a t e d with 1t/ (b(12, ~1.12 ~ ( [ 0.:17. T h e for~uvl:~t.ed ~tilml)ti(le was purified by l~igh-voltage p a p e r electro° phore.-is.
,Y,y,t hcs is of N-,cct ylm.et h io, yl-~.um rtic acid ~.-;kspart.ic aci,l ~til)~.~zvt ester p-t.olucne suit'(u-mr(', was sy~t~hesized a c c o r d i n g to G r e e n stei~ an~l \Vi~itz ( [ g i i l ) . This ('ontl~(~um.l a n d N - a c e l , y h n e t h i o u i a e r e v e a l e d t h e d e s i r e d bl+)ckt.~l lml)ti~h • I~v+al)l)lir~ation of t.h,~ t'n rl+()diimid, P met.hud (Gre.enstcitt a n d \Vit,itz, 1!t61). . , A f t e r l,vdr()g~uati,.~ with 10~/o '].d/C,"' c a t i o n e x c h a n g e c h r o m a t o g r a p h y cm A m b e r l i t e l ll-12(I ( l I " f~,vln) w'~n ]mri'r~rtned. T h e fractio~t o b t a i n e d aftxw e l u t i o n w i t h w a t e r was ('olh.ct,,([ am.[ lyOl)hilizcd. 'l'J,is prel).,ratio,~ c()ntailte([ l n u c h N - a c e t y l - M e t a n d ab least ~ , u,~id~ntilied c,)mpout~d. .V-A,t.tylln~qhio~in(, was l)ur(qmsed f r o m E. Mer(:k A(_I, D a r m s t a d t (No. 5(10984).
Pro~m.¢e di.qrsthm (.)l~f: 7r:~in ,,f ~.-(.ry:atallill was di.~s,,lved in 300 ml of w a t e r v i m t h e p]-[ v a l u e o f thLs s(~lut i~,l~ ndjuMe([ I,~ 8-0. AI~ (~Itzs'llle soll.tl;iolt c o n t a i ~ i n g 20 m g p r o n a s e P (Serva, t I e i d e l b,,r.,_,: t'v~,~n Strept.~y,:es grixeux) in 2 ml (1.(15 5~ NI[aHC0:~4-0"002 5I CaCl., w a s all(led. T h e p l l - f th,. n~ixturc was k e p t a t 8<1 ;It 3 7 ° 0 by ad~lition of 0.2 N NaOIY, u s i n g a R a d i o m e t e r l~l]-sl;,t ,ss(.u~l~lv. Aftor a p p r o x i m a t e l y 2} h r t.he alkali c o n s u m p t i o n was abou~ 1.30 i~tn(,I(,s a~,l tttt, u b e c a l m ' ncgli.~ibl~., l ) i g c s t i o n w a s e c . n t i n u e d for a n o t h e r ½ h r "rod w,.~ f,,li,)wm[ by lyophiliz'.~tio~.
ls,datio~r c,J tdr,(:/,'ed pel,tide by itm exch(rngc '['h~: Ivophilized. p r o m i s e digesti(,n mixt.ure f r o m l_ ~, o f ¢z-crvstallin. w a s d i s s o l v e d in 8 m l t,f w a t e r a n d a p p l i e d t,u :t D(~wex 5 0 \ V - X 2 c o l u m n ( 1 . 2 x 9 0 era). T h e eat, ion e x c h a n g e r~.silt was in th(, Ill + form a n d ]tad b c e a w a s h e d w i t h w a t e r u n t i l n e u t r a l . A f t e r e l u t i o n ~ d t h ;val er. ;~bou t, 10,5 o f t h e p e p t i d e m i x t u r o was not. r e t a i n e d b y t h e ion e x c h a n g e r . T h i s b l o c k e d p,.t.~t i,lc nm terial was l y o p h i l i z c d .
ll ~)/h-rol/
C~.rbos!lj~el,tid<,se tre(~tme'nt of the blocked dipe.l~tide T w o milliliters of 2 ~ N H f l - I C 0 3 a n d 0-5 m l of e n z w n e s o l u t i o n , c o n t a i n i n g a b o u t 1 m g of dS-isopropyl f l u o r o p h o s l ? h a t e - t r e a t e d c a r b o x y p e p t h t a s e A, were a d d e d to a p p r o x i m a t e l y 0.5 m g of t h e M o c k e d d i p e p t i d e in 2 mI of w a t e r . T h e e n z y m e w a s p u r c h a s e d f r o m S i g m a a.s ~, s u s p e n s i o n , c o n t a i n h l g 50 m g / m l ; it. w a s w a s h e 4 w i t h cold w a t e r a n d d i s s o l v e d in Water l~y a d d i n g t r i e t h y l a m i u e . T h i s s o l u t i o n was a d j u s t e d i m m e d i a t e l y to p K 8-0 b y a d d i n g 0-2 x a c e t i c acid. H y d r o l y s i s was p e r f o r m e d a t 37°C a n d t e r m i n a t e d b y a c i d i f y i n g w i t h glacial a c e t i c a c i 4 to pI~ 3. A l i q u o t s of 0-75 ml w e r e r e m o v e d at. 0, 10, 30, 60, 120 m [ n a n d 24 hr, l y o p h i l i z e d a n d e × a m i n e d b y t hi~t l a y e r c h r o m a t o g r a p h y .
29.t
Jl..).
liO],;XDI:;II,S I.;'P ski,
7'hi~ hz!ter chrom.alO!lraph.q I)C .[~'ert.igplatteu Ki(:selgel .F.,.w~ :~0 :-. 20 r.m (!']. M~w('k AG, I ) ~ r n ~ s t a d t ; N , , . - ) 7 1 5 ) w e r e a e t . i v a t e d b y .heat;i~g at, ! [IP~C f,,r / hv ;~,~t st,,wed in ;~ <[essicaf.or c,)ni,aining silim, gel ;~s , h ' y i n g a g e n t . Asct, nd.ing d c v e l o l ) m e n t r,f bite c h r o , n a t . , ~ g r a m wit& chl,,r, f f o r m n u ' t h : ~ , , l . } 1 7 ' yo a llitn, mi~ (g@-2,-}-I I)y w d u l n t , ) v,:quired rib,nil, ~-5 h r flu' 1i5 ,'m migrat.i,m ,d' th,. s, dv(..nb f r ( m t . A f t e r (~x'ap(~r:t~.ing ~t" I,h~." s o l v e n t (3~1 inin a~ l(l()°(J) tim plat.(..s w(,r,, statim',l. x~'it]l (~it.]Im" l.he nin]~ydrit)/(..JtG ~ re:,genl, (M,)ffat. a n d l,yt.le. I'qr)!)) (,r t'~)r ~l,'le('ti,m ,~f t.lm conlpoltn~[s wi(:h a b l o c k e d a m i n o gr(ml) w i t h l h e It.(,in(h.~l-lit,p|m rea~enl, a(:c~ "~|in.~ t.o y o u A r x atnd N e h e r (.1.qlL/).
A'm ino acid a nal/l.s'ix P e p t i d e s a m p l e s w e r e hydr,,'..vzed i~ li x I1(?1 '~t. 11()~C for lfi In'. Th,. subs,o,
(~~".I h r al. I~0'(? wil.h ~he "~id ,~f:2 .,," }J('l-mvtl~.:nol. :k P a c k a r d i,~sgrum,;nt (Series 7800) ,'quil,p,.d wi~h a hs, dr<,gen flam,r i,mizati,u~ d , ' l , ' c t ~ r w a s used. i t was [mssible to d e m , m s t . r a g e t h e pr,,s,.~ce ~,t" ~net.hv} acet.~to in exc,.ss , , f 2 N l t C l - m e t l m , ~ , } ,m a 181.1 ;. ().,t < q n i~oral,ak Q ,'olu~fi~ "~b l(ill'~C w i t h nit.roge~ as ,:am'i,_,r ~a.s.
.\'.uclear .ma.(l~telic re.~omt~Jee .,:peetrox(:o]~/ T h e lO0 M e / s prot.on n m g n e t i e re.~,,nun,.'e sp~.clra ~,f ~V-fornl.vl-.Met-Asl~ ',lut ,,f Ill,. A'i,cylnt~.d dipept.idl', o b t a i n e d f r o m ~.-t,rystallin. d i s s o l v e d in I).,O, wore r u n ,m ;~ V--,rian ttA-10() s p e . c t r m n e t r . r al. 35
Uttraeentr~J'ugal a n,d.qsis T h e p u r i t y o f ~.-,.vysl,allJn p r o p a r a l , i,ms was c o n t r o l l e d b y s e d i m e n t a t . i o n vel,~r.itv ruT,s in a S p i n c o m o d e l ]~ a n a l y t i c a l u l t r a c e n t , ' i f u K e , usin,~ t h e s,,1,1ie,'en o p t i c a l s y s t e m . I~t,,t;:in c o n e e n t . r a t i o n s we,'e 8 - 1 0 m g / m l . 'l.'l,e solvel0~ was 0-15 51 Na("t @0-02 5~ phosphaI.,., pl:I 7-3.
Qua.,ntilal.ire e.~ti.matio~ of ,teetgl 9 r o u p s D r y c,.-crystallin was s u b m i t . t e d t.o l~),drolysis in 2 ~" H O l - m c t h a n o l (ab,~nt 25 m g / m l ) d u r i n g 16 h r a t 100°C. T h e p r o c e d u r e describe,1 l)y I m ( l o w i e g a n d ] ) o r f m a n (196[)) wa.~ a p p l i e d t,) e s t i m a t e t h e a c e t y l c o n t e n t . T h e c a l i b r n t i o n o f tile m e t h o d w~Js perik~rmed wit.h s t a n d a r d s o l u t i o n s in tile c o n t e n t . r a t i o n r a n g e o f 0 - 5 / ~ m o l c e t h y l a c e t n t e pot- m i l l i l i t e r o f m e t h a n o l - u ' a t e r (1 + ] by vohz:ne.). :k ]ine,~r r e l ~ t i o n s h i p b e t w e e n opt:lea] de~,sit-y a n d ~ceta.te coneentrat.ion was obtained.
3. R e s u l l s ~-Crystallin ICrom 50 c a l f l e n s e s 1-5 g ~ . - c r y s t a l l i n w a s o b t a i n e d a.s d e s c r i b e d u n , l e r ,%{ctltods. T h e p u r i t y o f t h e p r o t e i n w a s j u d g e d b y s e d i m e n t a t i o n anal3~sis. P l a t e 1 s h o w s t,hat~ vhe g e l filt.ra~ion s t e p is n e c e s s a r y in o r d e r t o r e m o v e m a t e r i a l , s e d i m e : - , t i n g s l o w e r t.han t h e ma.in f r a c t i o n ( a p p r o x i m a t e l y 1.7 s).
IO
~D
'~)
•
:..:
:
~'i':,i i~i
'
.:
,
!
f
t. .
.
.
.
:!i:;111 : :fill
: ~.:;,,
.
.
!:111 ~
,
]?LATI.]J.. ultracentrifuge patLerns of ~-crystallin at 207C and 59,780 rev/min. Cancentvation approximately S nlg/mt. Sedimcntaionis to the left. (a) Witlmut the final gel filtrat,ion step in the preparat.ion procedtu'e: 10, 14, 18, 2'2 and 26 rain after reaching maximum speed. {b) Gel ftltratmn on 8eplmdcx (,-.~00 induded: 17, 19, 21, 23 amt. 25 rain after reaching maximum speed.
'~
!;I
7ao
2~
296
II:,
J,
I[OEN'I)
I,]J:IS
Ig'l?
AL
[ h c n a t u r e of l.he l)locking ~rOUl) w a s um'a.veled by tim following methods: (a.) Carbohydrat(; dM no't seem to l)e involve([ because of n e g a t i v e anlll r, u m react i()~t. (b) Comparison on high-voltnge p a p e r eh?('t.rol)horesis of t.he dil)el,ti~h: wilh swlthetic N - f ( , r m ) ' l - M e t - A s l ) a n d N-acctyl-51et-As]) gave nn indicali(m that. l he bl(,('king ~ ' o u p was ace tyl. '.l'he m i g r a t i m t distances were o n l y s l i g h t l y different. I)et.wet'~t the two s y n t l l e t i c (tipel)tide.s and t.he hlo(.'.ke(! dil)el)l.itle (Fig. 2). q'lu. same was trm, for the ninhy, lrin-m.,g;Ltivc lmrt of l.he d i p e p t i d e , wl~icll was released a hm" c.arboxypel> tidas(', t r e a t m e n t . The ]~I valtt(: (if N-form)d-Met. was lower lha~t t h a t ot' lira ])lock(.(l met.hioniEe r,.,leased from the pepti(h: b u t a.gain t;he ([itt'erem',e w~s sm;dl (Fig. :~).
® /V-FormylMet-P~sp..
(:
":
Blocked
N-Ace'~yl
dipeplide
',~ el-Asp
•: : :....::"
.::.7::. -
|C
-Mef z.:::::.;
U
g, E"
..°
|
'=
L
,
~
,
'g'"
:d'
.
,
'~D - > - -
® 1"1(:. 2. I[igh-v,Htage pap(.r electr,)l)h,)re.-.i.~ at, p l l 6-5. A. N-Formyl-.Met-Asl); Jl, .\'-acct.vl-Met-Asp; C. bh)vkcd (lipepti,le; |). N-a,.:etyl-Slct-AslL c , , n t a m inated w i t h A'-acetyl-Met a n d an unirlentifi('d c~,lnl)~,nent. R e i n d e l - I [~)l)pc r e a g e n t used l;.u-st4lining.
(c) Gas c h r o m a t o g r a l ) h y of t.he h y d r o l y z e d dil)eptide r e v e a l e d t . h c occurrence of an acet, y] group by t.he u.l:,l)earance of a. m e t h y l a c e t a t e peal¢ (Fig. 4). The sa,ne met hod h as r e c e n t l y been a.pplied by de \Vitt a n d I n g r a i n (1967) for t:he d e t e c t i o n of ac,:,tyl ~ - o u p s in bullfrog hemoglobins. (d) N u c l e a r m a g n e t i c resormatce (NStR) s p e c t r o s c o p y con firm cd t he gas el lrozn:~t:og r a p h i c finding. We com])arcd sxqlthct ic N-formyl-~lcb-Asp w i t h our blocked dipel)tide. The spect.ra are s u m m a r i z e d in T a b l e I. The s u b s t a n c e of n a t u r a l origirt lacks the f o r m y l p r o t o n signal (8 = 8-12) arm instead shows an a d d i t i o n a l m e t h y l 1)eak (8 = 2-04). Analogous c h e m i c a l shifts were r e p o r t e d for N , N - d i m e t h y l f o r m a m i d c (8 = 8-02) a n d for L r , N - d i m e t h y l a c e t a m i d e (8 = 2-08) (Bhacca, J o h n s o n a n d Shoolery, 1962). I n conclusion t h e N M R s p e c t r a s t r o n g l y i n d i c a t e thc s t r u c t u r e s ([) a n d ( I i ) (Table I) a n d i)rovc the presence of an a c c t a m i d e fra~qncnt in (II).
N-'I'EI~I]:NUS
;':.::
¢--:::
O
¢:::::
O Lr ~ - { ] ] { & " S T A T , ] , ] N
297
AcelylFormyl- Met M e.t. , .
"~
M e t :o"o.,
,:::?.:
:._...o ..... ,
B
C
,asp .... •"...i
,::_ :~-:> A
C::
B
<;£" A
.
D
C
B
I'~1(~. 3. Thin layer ( : t t r o m a t o g r a p h y (,T. silic~ gel. A, Carb(~xy])el)tidase (tiges(, (24 lit) of the I)loeke(t (LipcI)tide; ]g, N-ac,'ebyl-SIct; C, N - f o r m y l - 5 [ e t ; D, Asp ~l.nd M(~t.. Reind(H-Jl'ol)pe~ r~-agcat, usrd for staining.
Methonol
\ ~
~
~elhyloce~me
H20 HCL 0
5
~0
15
Time (rain) 1~'Io. 4. Gas e h r o m a t o g r , l m o b t a i n e d on ;~ l~orapak Q c o l u m n a t 160 °. T h e blocked dipeptide was h y d r o l y z e d in 2 ~ H C l - m o t h a n o ] . A n ak;.quot o f 1 #1 was i n j e c t e d into the colunm.
298
lI.
J.
HOlgaNDEIIS
E:I'
A].
Acetyl grou2)s in ~-cr!/stall.in The a c e t y l content, of tim ilml.(:t p r o t e i n was de+,.e.rnfiJmd by the m e t h o d of Ludowieg and D o r f m a n (1960). -k ratio of ram acetyl group per 21,000--.1=.900 g of p r o t e i n was calculated from duplic, ate experiments. TA 'ram 1:
Nuclear magnetic rcsona~w(: ~')mctra of .N-for,~yl-31el:.'Is)~ aml the blocked dipvytidc N-l:ormyl-Met-Asp {1) ~- Va hl/: J ' a r t m'.rt
8"12 -I -(i 4-5 2"60 2-10 2"05
,%ingl~'t [.(u:k [)cat" p a t t ('z'n B r u i t iph.} I),,ubh't T r i pier. Singlet (h,n,plex
--
---
2.92
1";27
] H o c k e d (li 1. t'l,ti(l,: (I I ) 8-X'alm, Patter,,
.:\ssignmcftt
,t I, ,.
. . . . . . . . . -l-G !a~('l¢ b e a t l m l t e r n 4-.', 31 u h.i ph-l. " 2"1t".2" I.h ml*b:t 2.{it ) T r i plot 2-I0 Sin~lel 2-1)5 (.'-,m, p h ' x 2-0 [ SiJ'tglot . . . . . .
d
. f r.,, L
3htltiplet
iu:Imri~5 .
.
.
I
l Cl[.e
}
I
{
Ii t
0
H b
I
It
l-[ () c
!
tI
f
('IL_ 9
CI{= d
(I) 1 - [ - - C 2 - - . N - - ( J - - - C - - - N - - C ~ - ( ' O O I
{ ' ( ) ( ) | [ I:
1
I
C'H= .q
-
('1l._ ,~
C0()11 b
I, (1[)
[ !,
l,
("lla---(.{
I}
i
t.'l'l._, d -
I
...... N . . . . ( 7 . - - - ( ' - - - . N - .
i
:
1,
g
!:
A'.--G(H
)II L
i
]t c
c
F i r s t - o r d e r c h e m i c a l shift.-, a r e g i v e n .'t-; 3 - v a l n e s . Lakintz 5 == 2-10 fi,r t h e - - . S - . - . ( ' l l ~ p r o t , m , . r e l a t i v e i n t c n s t ~ , i e s a n d t h e . i n d i c a d e d t a ~ l t i p l i c i t i e s c.ozttirm the. a s e i g m n ( . n t s .
4.
"Yh,"
lSiseussion
For a n u m b e r of years the N-termirum of ~-crystallin remained an mtsolvec[ probiem. Alt.hough D N P - g l u t a m i c aehi was found w i t k Sanger's FlSNl-~-nmthod, t.he a m o u n t .of N - t e r m i n a l g h t t a m i c acid ~H)peared to be too small to be. sigxfifieant, in view of our knowledge of-the s u b s t r u c t u r e of y.-crystallin. ]3life, trait i n t e r p r e t a t i o n s of the discrepancy between the q u a l t t i t y of N-rorminal a m i n o acid a w l t.}m molecular weight of the sltbltnit-s were pos~sible: (a) The architectm'c of ~-cry.stalli~t cotfld be such t h a t tile N-termin'al a m i a o acid was a t least p a r t l y inaccessible. (b) ~f glutsamic acid was t h e N--terminal a m i n o acid the f o r m a t i o n of pyrr61id-2one-5-carboxylie acid nfight h a v e t a k e n place, t~ing closure, of this t y p e has been reported, e.g. b y Press, tSlggot and P o r t e r (1966). (c) C a r b o h y d r a t e could mask t h e end group.
N-TFIP,.M:INUS OF ~ - C I ~ Y S T A L L I N
299
(d) A c v h t t i o n ~:rf" t h e ~-Ln~ino e n d g r o u p c o u l d l ) r e v e n t c o u p l i n g w i t h li'DN]3. T h e l a t t e r p o s s i b i l i t y w a s l?OStttla.tcd b y ] 3 ] o e m c n d a t c t al. (1965). ( A d a) A s r e p o r t e d earlim" b y B l o e m e n d a l et, al. (1966), n o i n c r e a s e o f N - t e r m i n a l g h t t a . m i e a e M w a s o b s e r v e d w h e n t h e c o u p l i D g react, i o n wit;h F D N B w a s p e r f o r m e d in t l m p r e s e n c e o1" 7 .~t u r e a . T h c ' r e f o r e , p o s s i b i l i t y (a) c o u l d b e e x c l u d e d . (A~t b) A m t m b e r o f e x p e r i m e n t s w e r e p e r f o r m e d u u d e r co/tditio~ts t h a t r e s u l t e d in o p e n i n g o f t h e p3n'rolidonc: r i n g ; h o w e v e r , a h i g h e r a m o u n t o f N - t e r m i n a l g h m r m i e aci(:l c o u b t n o t I)e. d e t e e t e c l . ( A d ~,) ( ' a r l , o h y d r a t e negative.
gmmps couM
be r u l e d
o u t as t h e a n t h r o n e
reaction
was
(A,I d) As m e n t i o m : d Crarlier (/31ocrmmulal eL a.]. 1966), t i m p r e s e n c e o f ,~Lf o r m y l o r a,n a(,~.t yl g , ' - u p w a s v e r y ] i k e l y . h t t i m r e m t l t s s e c t i o n wc h a v e n o w d e m o n s t . r a t e d ~'it.h o ' , ' e r w h e h ~ d n g ex'i~lence t h a t t.he b l o e k i ~ g gq'oup is a c e t y l wl~ile t h e N - t e r m i n a l a m i i m a c i d is ~,wtl,~mi~m. T i m s e c o n d a m i n o a c i d a t t h e t e r m i n u s w a s f o u n d t o be a s p a r t i c acid. Fm'th,n" i u v e s t i g a t i m t s a r e i n progq'ess in o r d e r t o a n a l y z e t h e s m a l l a m o m l t o f or.her b l o c k e d l ml)ti
.
'
•
A CK X 0 W L E ]3 0 M E N'1:8 \V;, s h o v l d like t,5 thau.k Profe.~sor J. \V. lg. C o e n e n an<[. Mr M. v a n O o r k o m fro:. t h e porl'~n'manc:i:' of N3iI{ specg'roscopy a nd in t;erpregal~ion of tJte results. T h a n k s are d u e also t o Miss .J,do: v a n Tol, Mr' A. K o p p e r s a n d Mr J. R,e i j n e n for t h e i r skillecl t e c h n i c a l a s s i s t a n c e . P,.E:F151-~ E N C E S
yon .'\rx. E. :rod Ncher. ]~.. (1963). J. Chro~nalog. 1 2 , 329. ]3haeea, N..%.'.. john.,son, L. :F. and S.hooler3". 3. N.'(1962). Varian £'pectra C'atalogue, spectr~ 39 resln-.ctively 42,1..N~ttional Press. U.S.A. t31ocmendal. H. (1967). Thesis Univcrsit.y of A m s t e r d a m . .13loemendal; }I.. Bont., \V. S., ]3enedetti. ]g. I,. a n d \Vis.6c, .1. I-[. (1965). ]'2xTtl Eye Res. 4, 319. T~loemend~d. 1{. a n d ten (;ate, O. (1958)..Vature 1 8 1 , 340. ]31ocrnendM, 14.. Bo~3t, "~\:. S., J o n k i n d . J. F. mid %Zisse, J . I - L (1962). E~pll Eye _Re~. 1, 300. J31oemendal. 1;I., ]foenders, H. J., 8ehocnmakm~, . J . G . G . , Zwee~, _-k.a n d 0erding, J. J. T. (1966). l~roc. Syrup. d~io,'hem, l'~!/e (~_I'utzing, Gcrmal~y) (in press): van l ) a m . A. 1v. a.nd ten Care, G. (1966). ]3iochim. Biol)hy.s. Act(t. 121, 183. Firfarova. 1-2. I v. (1958). Biol:himiya 2 3 , 129. Gra,s,smann, '~V. and ~Viinsch, E. (1.95S). Chem. Bet. 91, 449. Greenslein. J. P. and ~Vinit.z. 3[. (i961). Chemistrfi of the A m i n o AcicL~, p. 942, 1016. J o h n Wiley &. Sons, New k'ork ~md London. ]:{ahnel, R . (1961). M rch. OphlhalmgL 163, 283. ]:[oenders, }[. J. and J~loo.nmnda.l, J:{. (1967). ]3iochDn. ]31ophys. Act(~ 147, 183. Ludowieg, ,J. a n d ])orfman, A. (19{J0). J3iochDn. Bioph!/s. A cla 38, 212. Moil'at., E. 19. and Lytle, P,. I. (1959). M nalyt. Chem. 3 1 , 9 2 0 .
300
H. J. t [ O F , N D E R , ~ E T AI,
])re~,;. E. M., l?it
N-Terminus of m-Crystallin FI. ,J. ]Iot.:~m.:~s, 3. O. G. SCHOV;X,~AKmtS, J. J. 'l?. C,~.~r~D~r~c,, G. I. T~.:SSI~R* .~.~t) l I. BLO~++~J..:m)At,
Department of Biochemistry, University of Nijmegen, N0",te.qen , The Netherla.nds (Pape, f,om the Symposh+m.
Lc,es Proteins and Relatexl Subjects", Ghent, Belgium, June 1967)
:k p u r e N - t e r m i n a l p('pt.i(I¢: wa,,+ i~olal:(.-d f r o m b o v i n e l e n s ;¢.-crystallill ,~,l+ter prono, se d i g e s i i n n , eat.ion e x c h t t n ~ e on l ) o w e x 50~V-X2 ~uxd t w o atldibiom~l e l e c t r o l ) h o r e t i c purific~t, iott s t e p s . :['he c ! ! w i d a l i o n o f l.he sl, r u c g u r e b y m e a n s o f h i g h volg~+ge eleel~rophoresis, t h i n l a y e r e | u ' o m a u o g r ~ p h y , g a s c h r o m ' t t ( ~ g r ~ l ~ h y tutd. mtcle0,r magn.et, ic rosotl~xrtce speot, r o s e o p y r e s u l t e d in t h e t'ormul~v: z'V-acct,yl-3[el,-Asp. <)~t~; a('+,tyl grotl|) xvtis foull(! p e r a p p r < ) x i m a t c l 5' 2 1 , 0 0 0 g o f ~ - c r y s t M l i n . T h e l a t t e r v a l u e is t-~n~sistent, w i t h tht~ mt~h.cttlar w e i g h t o f ghe s n b m x i t s . 'l'ht~ p r e v i o u s f i n d i n g s t h a e g l u t . ~ m i e a c i d w a s t h e N-t
i. Introduction Since 1957 a mtml)er of laboratories rcl)orted t.lu~t glut, amic acid was t h e N - t e r m i n a l arnino aci~l of bovim, le,~s ~-erys.tallin (].~loemendal, 1957; B l o e m e n d a l and t e n Cate, 1958; ]°irfarova. ] 958; H/thncl, ]96]; v~,t .Dam a,nd ten Care, 1966). I,t r('ecld: ve:~rs the (le-q'ec of ]mrit.y of a.-cryst.,dlill p r e p a r a t i o n s has been iml,roved by lneans of elcetrophoresis, d e n s i t y g r a d i e n t centrifugation, p r e c i p i t a t i o n techniques and ,gel tilt:ration. I n t;hesc highly purified a.-crystallin :preparations o,gy one inch. of N - t e r m i n a l g l u t a m i c acid per a p l ) r o x i m a t e l y 2.]06 g of p r o t e i n could be detected (Blo,,me,ula], B e n t and Be,mdctti, 1965; Bloemendal, .I-Ioenders, Schoenmakers, Zwecrs altd Gerding, 1966). A p p a r e n t l y the larger a m o t m t s of N - t e r m i n a l g l u l a m i c acid prcviolmly observed were p r o b a b l y caused by cmlt,~n~ination, sitme practically a.II of it co~dd be removed, especially by gel filtration. ]31oemendal, ]3ont, J o n g k i n d and Wisse (1962) dcmonstrz~ted t h a t 0¢-crystallin (molecular weight approximate.]y 800,000) possessed a q u a t e r n a r y structure and t h a t it could be reversibly dissociated into sttbunits or p o l y p e p t i d e chains with a molecular weight of a b o u t 25,000. '+['hlus for cach s u b m d t the amouul5 of D N P - g l u t a m i c acid present is h a r d l y siguiticant and could be desigmtted as a trace end group. One e x p l a n a t i o n of this curious fact is t h a t tim N - t e r m i n a l anlino acid of ~.-crystallin was inaccessible to t h e reactiotx with 1-fluoro-2, 4-dirficrobenzene (:FDxNrB). The possibility t h a t t h e N t e r m i n a l a m i n o acid was blocked by a c a r b o h y d r a t e or an acetyl group was suggested by Bloemendal et al. (1965). I n a symposium, ~,Valey (1966) eommmxieated t h a t q u a l i t a t i v e analysis of pronase digests of c¢-eITstMli~l showed the presence o£ a blocked t e t r a p e p t i d e with m e t h i o n b l e as the N - t e r m i n a l amino acid. We d e t e c t e d a blocked dipeptide in digests of v.-crystallin in n u m e r o u s ex~erimelxts. I n t h e p r e s e n t paper vim composition of the dipeptide and the n a t u r e of t h e blocking g~'oup recovered in c¢-erystallin digests is reported. A p r e l i m i n a r y a c c o u n t has been given elsewhere (Iq:oenders a n d :Bloemendal, 1967). ~<1 3 e p a r t m c n t o f Organic C h e m i s t r y , U n i v e r s i t y o f -.-N~!jmcgcn, N i j m e g e n , T h e N e t h e r l a n d s . 291
•2q2
1l. J . H O.I':XDF. IIS I'.'T AL
2. ~ i a t e r i a l s and N I e t h o d s
Prej~aration. of lens extract Calf e y e s were ol)tailm~l f r e s h f r o m the s/aught.erhouse a n d the lenses were r e m o v e d i r a , h e & l a t e l y F i v e milliliters of w a t e r were a d d e d to l g ot" It.uses. T h e war, or-soluble protein.s were e x t r a c t e d i~l a cold room by g e n t l e s t i r r i n g until t:he c o r t e x was dissolved. T h e I.m-bid s o l u t i o n was c e n t r i f u g e d fur 10-15 rain at. 25,01X):.: g a t a low tmnlmrat.ure. T h e r e s u l t i n g c l e a r s o l u t i o n was l y o p h i l i z e d a n d s t o r e d a t --25°C.
Isolation o.f c,.-ctT/stalli.n T h e m e t h o d d e s c r i b e d by S p e c t o r (19114), with s o m e modiiicat.ions, was used as the lirst p u r i f i c a t i o n step. All m a n i p u l : ) t i o n s were 1)evformcd at. 0-5~'C.. One millilit.ev of 0.1 .~1 '['ris buffer (pi'[ 7-3) a n d 60 i~1 of t).173 5t zinc a c e t a t e , dissolved in the s a m e buffer were a d d e d to 5 ml of t h e lens e x t r a c t . A f t e r I5 rain, 0.9 In[ o( iec-coi¢[ e t h a n o l was a d d e d slow])" to t h e solution w i t h stirring. A f t e r centrifllg;ttion ab 25,0007.,g the SUl)ern:ttant fluid wa.¢ brought, to pI-I 6-5 w i t h x ItCL T h e i)recipi~ate was r e n m v c d by (,(.nrcifugation a n d t h e s u p e r n a t a n t fluid acidified w.t.th x HCI t,, tim is~mh:ct.ric p o i n t of a.-e,'ystalli~t (pl-[ 5-2). .Fbaal/v, tile p r e c i p i t a t e d m a t e r i a l was s u b m i t t e d to gel filt.rabi
Sy~ thes is of N -formyl-L- rneth io nyl~ L- aspa rt ic acid (a) Synthe-sis o/ N-formyl-t.-methionyl-r-aslmrtic ~tcid ,limeth?tl e,ster, 2"0 g (10 m n m l c s ) of L-aspart.ie a c i d d i m e t h v l e s t e r hy,'dvochh)ride ((]rnssnt;Lnn aml \Viins('h, 1958) were dis.~olved in 5 ml of d J ~ n e t h y l f o r m a m i d e a n d ([iluted. w i t h I0 nd ~f e t h y l acctat.e. '.!2o t h e nlixtu.re 1-4 ml (10 mmoles) of t r i e t h y l a m i n e w e r e a d d e d a~M the whit.e l)recipit:at#, was filtered off a f t e r 5 rain. To t h e filtrate 1.8 g (1() m m o l e a ) N-formyl-u-methi,:~ltine. ( W i n d u s a n d Marvel, 1931) were a d d e d imme,.t a t e l y to p r e v e n t i n t e r n a l a n d i u t r a m o l e c u l ' a ' cond e n s a t i o n s of t h e e s t e r t'uncGons du(*. to t.he I)rese'we .... )f .~ free amino, group, a n d 2"..'3 .g (20 m m o l e s ) N - h y d r o x y s u c c i n i r n i d e (Wiin.-:ch a n d J a e g c ' , 1966) were dissolved in t h e m i x t u r e ' ( t h i s s n b s t a n e e in.hibit,a r a c e m i z a t i o n a n d o f t e n i m p r o v e s the yield in pept.itle couplings w i t h dicyclohexylcarbodiimi(1,'.% \ V e y g a n d , IIofl'nmim a n d Wiinsch, 1966). T h e m i x t u r e w a s t h e n cooled bo ---22~'C a n d a solution of 2-20 g (10-7 mmoles) dicycl<,h e x v l c a r b o d J i m i d c in e t h y l a c e t a t e was a d d e d . The r e a c t i o n flask was k e p t "tl; this t e m p e r a t u r e for 15 rain a n d t h e n left ove/'night, in t.he refi'igeru~or a t 0°0. T h e n e x t d a y , precipit a t e d d i c y c l o h e x y l m ' c a was filtered off nnd, t.he residue was w a s h e d w i t h a s m a l l a m o u n t o f e t h y l ncet.ate. ~ h . flit.rate was c o n c e n t r a t e d in v a e u o a n d d.ilutetl w i t h c h l o r o f o r m . T h e s o l u t i o n was t h e n t,'ansferred to q s e p a r a t o r v f m m e l a n d w a s h e d s u c c e s s i v e l y with wat.er, s o d i u m b i c a r b o n a t e a n d citric :mid solutioJt a n d again with w a t e r ( a d d i t i o n of s o d i u m c h l o r i d e to t h e s e e x t r a c t i o n solutions will p r e v e n t th('. escape of a n y of the w a t e r - s o l u b l e product.s). T h e e]xloroform e x t r n e t wns d r i e d wit.h s o d i u r a s u l f a t e a n d e v a p o r a t e d t.o leave 9-.50 ~ (78~/o) or a colorless oil. T h e m a t e r i a l was used as s u c h ia t h e nexl, step. (b) Hydrolysis of the protected dipeptide ester. K y d r o l y s i s was p e r f o r m e d a f t e r dissolving of t h e m a t e r i a l in w a t e r a t p H 12 b y a d d i t i o n of 4 x s o d i u m hydro.xide. T h e s o l u t i o n was k e p t a t t k i s pt-I v a h t e for 10 rain. A f t e r r e m o v a l o f a small a m o u n t of insoluble m a t e r i a l it w a s filtered t h r o u g h a c o l u m n of s t r o n g l y acidic ion e x c h d u g e r (Amberlite I1¢-I20). T h e c o l u n m w a s w a s h e d w i t h w a t e r u n t i l the initially acidic effluent ( p H 3 - 4 ) r e a c h e d n e u t r a lity. T h e effluent w a s lyopKilizecl to leave a w h i t e a n d britt.le f o a m (1-95 g -~ 85~/o). T h i s material k e p t a t r o o m t e m p e r a t u r e t,ransformed after s o m e d a y s to a hard glassy mass. T h e acid d i s s o l v e s easily in wat.er a n d in alcohols, b u t is insoluble in n o n - p o l a r solvent, s. T h e p r o d u c t .was s u b j e c t e d to t h i n l a v e r c h r o m a t o g r a p h y on silica gel using the s y s t e m e t h y l a c e t a t e + p y r i d i n e - l - , a c e t i c a c i d + w a t e r ( 6 2 . - F 2 1 + 6 + 1 1 , b y v o l m n e ) . T h e m a i n spot,
N-'I'EI[MINU'S
OF
Cz. C R Y S : I ' A 1 .
LIN
293
eo~npri.sing abmai . q 0 ~ (d" t h e n m t e r i a l , al)p(,ar(.d a t l@ 0.32, mirror s p o t s m i g r a t e d with 1t/ (b(12, ~1.12 ~ ( [ 0.:17. T h e for~uvl:~t.ed ~tilml)ti(le was purified by l~igh-voltage p a p e r electro° phore.-is.
,Y,y,t hcs is of N-,cct ylm.et h io, yl-~.um rtic acid ~.-;kspart.ic aci,l ~til)~.~zvt ester p-t.olucne suit'(u-mr(', was sy~t~hesized a c c o r d i n g to G r e e n stei~ an~l \Vi~itz ( [ g i i l ) . This ('ontl~(~um.l a n d N - a c e l , y h n e t h i o u i a e r e v e a l e d t h e d e s i r e d bl+)ckt.~l lml)ti~h • I~v+al)l)lir~ation of t.h,~ t'n rl+()diimid, P met.hud (Gre.enstcitt a n d \Vit,itz, 1!t61). . , A f t e r l,vdr()g~uati,.~ with 10~/o '].d/C,"' c a t i o n e x c h a n g e c h r o m a t o g r a p h y cm A m b e r l i t e l ll-12(I ( l I " f~,vln) w'~n ]mri'r~rtned. T h e fractio~t o b t a i n e d aftxw e l u t i o n w i t h w a t e r was ('olh.ct,,([ am.[ lyOl)hilizcd. 'l'J,is prel).,ratio,~ c()ntailte([ l n u c h N - a c e t y l - M e t a n d ab least ~ , u,~id~ntilied c,)mpout~d. .V-A,t.tylln~qhio~in(, was l)ur(qmsed f r o m E. Mer(:k A(_I, D a r m s t a d t (No. 5(10984).
Pro~m.¢e di.qrsthm (.)l~f: 7r:~in ,,f ~.-(.ry:atallill was di.~s,,lved in 300 ml of w a t e r v i m t h e p]-[ v a l u e o f thLs s(~lut i~,l~ ndjuMe([ I,~ 8-0. AI~ (~Itzs'llle soll.tl;iolt c o n t a i ~ i n g 20 m g p r o n a s e P (Serva, t I e i d e l b,,r.,_,: t'v~,~n Strept.~y,:es grixeux) in 2 ml (1.(15 5~ NI[aHC0:~4-0"002 5I CaCl., w a s all(led. T h e p l l - f th,. n~ixturc was k e p t a t 8<1 ;It 3 7 ° 0 by ad~lition of 0.2 N NaOIY, u s i n g a R a d i o m e t e r l~l]-sl;,t ,ss(.u~l~lv. Aftor a p p r o x i m a t e l y 2} h r t.he alkali c o n s u m p t i o n was abou~ 1.30 i~tn(,I(,s a~,l tttt, u b e c a l m ' ncgli.~ibl~., l ) i g c s t i o n w a s e c . n t i n u e d for a n o t h e r ½ h r "rod w,.~ f,,li,)wm[ by lyophiliz'.~tio~.
ls,datio~r c,J tdr,(:/,'ed pel,tide by itm exch(rngc '['h~: Ivophilized. p r o m i s e digesti(,n mixt.ure f r o m l_ ~, o f ¢z-crvstallin. w a s d i s s o l v e d in 8 m l t,f w a t e r a n d a p p l i e d t,u :t D(~wex 5 0 \ V - X 2 c o l u m n ( 1 . 2 x 9 0 era). T h e eat, ion e x c h a n g e r~.silt was in th(, Ill + form a n d ]tad b c e a w a s h e d w i t h w a t e r u n t i l n e u t r a l . A f t e r e l u t i o n ~ d t h ;val er. ;~bou t, 10,5 o f t h e p e p t i d e m i x t u r o was not. r e t a i n e d b y t h e ion e x c h a n g e r . T h i s b l o c k e d p,.t.~t i,lc nm terial was l y o p h i l i z c d .
ll ~)/h-rol/
C~.rbos!lj~el,tid<,se tre(~tme'nt of the blocked dipe.l~tide T w o milliliters of 2 ~ N H f l - I C 0 3 a n d 0-5 m l of e n z w n e s o l u t i o n , c o n t a i n i n g a b o u t 1 m g of dS-isopropyl f l u o r o p h o s l ? h a t e - t r e a t e d c a r b o x y p e p t h t a s e A, were a d d e d to a p p r o x i m a t e l y 0.5 m g of t h e M o c k e d d i p e p t i d e in 2 mI of w a t e r . T h e e n z y m e w a s p u r c h a s e d f r o m S i g m a a.s ~, s u s p e n s i o n , c o n t a i n h l g 50 m g / m l ; it. w a s w a s h e 4 w i t h cold w a t e r a n d d i s s o l v e d in Water l~y a d d i n g t r i e t h y l a m i u e . T h i s s o l u t i o n was a d j u s t e d i m m e d i a t e l y to p K 8-0 b y a d d i n g 0-2 x a c e t i c acid. H y d r o l y s i s was p e r f o r m e d a t 37°C a n d t e r m i n a t e d b y a c i d i f y i n g w i t h glacial a c e t i c a c i 4 to pI~ 3. A l i q u o t s of 0-75 ml w e r e r e m o v e d at. 0, 10, 30, 60, 120 m [ n a n d 24 hr, l y o p h i l i z e d a n d e × a m i n e d b y t hi~t l a y e r c h r o m a t o g r a p h y .
29.t
Jl..).
liO],;XDI:;II,S I.;'P ski,
7'hi~ hz!ter chrom.alO!lraph.q I)C .[~'ert.igplatteu Ki(:selgel .F.,.w~ :~0 :-. 20 r.m (!']. M~w('k AG, I ) ~ r n ~ s t a d t ; N , , . - ) 7 1 5 ) w e r e a e t . i v a t e d b y .heat;i~g at, ! [IP~C f,,r / hv ;~,~t st,,wed in ;~ <[essicaf.or c,)ni,aining silim, gel ;~s , h ' y i n g a g e n t . Asct, nd.ing d c v e l o l ) m e n t r,f bite c h r o , n a t . , ~ g r a m wit& chl,,r, f f o r m n u ' t h : ~ , , l . } 1 7 ' yo a llitn, mi~ (g@-2,-}-I I)y w d u l n t , ) v,:quired rib,nil, ~-5 h r flu' 1i5 ,'m migrat.i,m ,d' th,. s, dv(..nb f r ( m t . A f t e r (~x'ap(~r:t~.ing ~t" I,h~." s o l v e n t (3~1 inin a~ l(l()°(J) tim plat.(..s w(,r,, statim',l. x~'it]l (~it.]Im" l.he nin]~ydrit)/(..JtG ~ re:,genl, (M,)ffat. a n d l,yt.le. I'qr)!)) (,r t'~)r ~l,'le('ti,m ,~f t.lm conlpoltn~[s wi(:h a b l o c k e d a m i n o gr(ml) w i t h l h e It.(,in(h.~l-lit,p|m rea~enl, a(:c~ "~|in.~ t.o y o u A r x atnd N e h e r (.1.qlL/).
A'm ino acid a nal/l.s'ix P e p t i d e s a m p l e s w e r e hydr,,'..vzed i~ li x I1(?1 '~t. 11()~C for lfi In'. Th,. subs,o,
(~
.\'.uclear .ma.(l~telic re.~omt~Jee .,:peetrox(:o]~/ T h e lO0 M e / s prot.on n m g n e t i e re.~,,nun,.'e sp~.clra ~,f ~V-fornl.vl-.Met-Asl~ ',lut ,,f Ill,. A'i,cylnt~.d dipept.idl', o b t a i n e d f r o m ~.-t,rystallin. d i s s o l v e d in I).,O, wore r u n ,m ;~ V--,rian ttA-10() s p e . c t r m n e t r . r al. 35
Uttraeentr~J'ugal a n,d.qsis T h e p u r i t y o f ~.-,.vysl,allJn p r o p a r a l , i,ms was c o n t r o l l e d b y s e d i m e n t a t . i o n vel,~r.itv ruT,s in a S p i n c o m o d e l ]~ a n a l y t i c a l u l t r a c e n t , ' i f u K e , usin,~ t h e s,,1,1ie,'en o p t i c a l s y s t e m . I~t,,t;:in c o n e e n t . r a t i o n s we,'e 8 - 1 0 m g / m l . 'l.'l,e solvel0~ was 0-15 51 Na("t @0-02 5~ phosphaI.,., pl:I 7-3.
Qua.,ntilal.ire e.~ti.matio~ of ,teetgl 9 r o u p s D r y c,.-crystallin was s u b m i t . t e d t.o l~),drolysis in 2 ~" H O l - m c t h a n o l (ab,~nt 25 m g / m l ) d u r i n g 16 h r a t 100°C. T h e p r o c e d u r e describe,1 l)y I m ( l o w i e g a n d ] ) o r f m a n (196[)) wa.~ a p p l i e d t,) e s t i m a t e t h e a c e t y l c o n t e n t . T h e c a l i b r n t i o n o f tile m e t h o d w~Js perik~rmed wit.h s t a n d a r d s o l u t i o n s in tile c o n t e n t . r a t i o n r a n g e o f 0 - 5 / ~ m o l c e t h y l a c e t n t e pot- m i l l i l i t e r o f m e t h a n o l - u ' a t e r (1 + ] by vohz:ne.). :k ]ine,~r r e l ~ t i o n s h i p b e t w e e n opt:lea] de~,sit-y a n d ~ceta.te coneentrat.ion was obtained.
3. R e s u l l s ~-Crystallin ICrom 50 c a l f l e n s e s 1-5 g ~ . - c r y s t a l l i n w a s o b t a i n e d a.s d e s c r i b e d u n , l e r ,%{ctltods. T h e p u r i t y o f t h e p r o t e i n w a s j u d g e d b y s e d i m e n t a t i o n anal3~sis. P l a t e 1 s h o w s t,hat~ vhe g e l filt.ra~ion s t e p is n e c e s s a r y in o r d e r t o r e m o v e m a t e r i a l , s e d i m e : - , t i n g s l o w e r t.han t h e ma.in f r a c t i o n ( a p p r o x i m a t e l y 1.7 s).
IO
~D
'~)
•
:..:
:
~'i':,i i~i
'
.:
,
!
f
t. .
.
.
.
:!i:;111 : :fill
: ~.:;,,
.
.
!:111 ~
,
]?LATI.]J.. ultracentrifuge patLerns of ~-crystallin at 207C and 59,780 rev/min. Cancentvation approximately S nlg/mt. Sedimcntaionis to the left. (a) Witlmut the final gel filtrat,ion step in the preparat.ion procedtu'e: 10, 14, 18, 2'2 and 26 rain after reaching maximum speed. {b) Gel ftltratmn on 8eplmdcx (,-.~00 induded: 17, 19, 21, 23 amt. 25 rain after reaching maximum speed.
'~
!;I
7ao
2~
296
II:,
J,
I[OEN'I)
I,]J:IS
Ig'l?
AL
[ h c n a t u r e of l.he l)locking ~rOUl) w a s um'a.veled by tim following methods: (a.) Carbohydrat(; dM no't seem to l)e involve([ because of n e g a t i v e anlll r, u m react i()~t. (b) Comparison on high-voltnge p a p e r eh?('t.rol)horesis of t.he dil)el,ti~h: wilh swlthetic N - f ( , r m ) ' l - M e t - A s l ) a n d N-acctyl-51et-As]) gave nn indicali(m that. l he bl(,('king ~ ' o u p was ace tyl. '.l'he m i g r a t i m t distances were o n l y s l i g h t l y different. I)et.wet'~t the two s y n t l l e t i c (tipel)tide.s and t.he hlo(.'.ke(! dil)el)l.itle (Fig. 2). q'lu. same was trm, for the ninhy, lrin-m.,g;Ltivc lmrt of l.he d i p e p t i d e , wl~icll was released a hm" c.arboxypel> tidas(', t r e a t m e n t . The ]~I valtt(: (if N-form)d-Met. was lower lha~t t h a t ot' lira ])lock(.(l met.hioniEe r,.,leased from the pepti(h: b u t a.gain t;he ([itt'erem',e w~s sm;dl (Fig. :~).
® /V-FormylMet-P~sp..
(:
":
Blocked
N-Ace'~yl
dipeplide
',~ el-Asp
•: : :....::"
.::.7::. -
|C
-Mef z.:::::.;
U
g, E"
..°
|
'=
L
,
~
,
'g'"
:d'
.
,
'~D - > - -
® 1"1(:. 2. I[igh-v,Htage pap(.r electr,)l)h,)re.-.i.~ at, p l l 6-5. A. N-Formyl-.Met-Asl); Jl, .\'-acct.vl-Met-Asp; C. bh)vkcd (lipepti,le; |). N-a,.:etyl-Slct-AslL c , , n t a m inated w i t h A'-acetyl-Met a n d an unirlentifi('d c~,lnl)~,nent. R e i n d e l - I [~)l)pc r e a g e n t used l;.u-st4lining.
(c) Gas c h r o m a t o g r a l ) h y of t.he h y d r o l y z e d dil)eptide r e v e a l e d t . h c occurrence of an acet, y] group by t.he u.l:,l)earance of a. m e t h y l a c e t a t e peal¢ (Fig. 4). The sa,ne met hod h as r e c e n t l y been a.pplied by de \Vitt a n d I n g r a i n (1967) for t:he d e t e c t i o n of ac,:,tyl ~ - o u p s in bullfrog hemoglobins. (d) N u c l e a r m a g n e t i c resormatce (NStR) s p e c t r o s c o p y con firm cd t he gas el lrozn:~t:og r a p h i c finding. We com])arcd sxqlthct ic N-formyl-~lcb-Asp w i t h our blocked dipel)tide. The spect.ra are s u m m a r i z e d in T a b l e I. The s u b s t a n c e of n a t u r a l origirt lacks the f o r m y l p r o t o n signal (8 = 8-12) arm instead shows an a d d i t i o n a l m e t h y l 1)eak (8 = 2-04). Analogous c h e m i c a l shifts were r e p o r t e d for N , N - d i m e t h y l f o r m a m i d c (8 = 8-02) a n d for L r , N - d i m e t h y l a c e t a m i d e (8 = 2-08) (Bhacca, J o h n s o n a n d Shoolery, 1962). I n conclusion t h e N M R s p e c t r a s t r o n g l y i n d i c a t e thc s t r u c t u r e s ([) a n d ( I i ) (Table I) a n d i)rovc the presence of an a c c t a m i d e fra~qncnt in (II).
N-'I'EI~I]:NUS
;':.::
¢--:::
O
¢:::::
O Lr ~ - { ] ] { & " S T A T , ] , ] N
297
AcelylFormyl- Met M e.t. , .
"~
M e t :o"o.,
,:::?.:
:._...o ..... ,
B
C
,asp .... •"...i
,::_ :~-:> A
C::
B
<;£" A
.
D
C
B
I'~1(~. 3. Thin layer ( : t t r o m a t o g r a p h y (,T. silic~ gel. A, Carb(~xy])el)tidase (tiges(, (24 lit) of the I)loeke(t (LipcI)tide; ]g, N-ac,'ebyl-SIct; C, N - f o r m y l - 5 [ e t ; D, Asp ~l.nd M(~t.. Reind(H-Jl'ol)pe~ r~-agcat, usrd for staining.
Methonol
\ ~
~
~elhyloce~me
H20 HCL 0
5
~0
15
Time (rain) 1~'Io. 4. Gas e h r o m a t o g r , l m o b t a i n e d on ;~ l~orapak Q c o l u m n a t 160 °. T h e blocked dipeptide was h y d r o l y z e d in 2 ~ H C l - m o t h a n o ] . A n ak;.quot o f 1 #1 was i n j e c t e d into the colunm.
298
lI.
J.
HOlgaNDEIIS
E:I'
A].
Acetyl grou2)s in ~-cr!/stall.in The a c e t y l content, of tim ilml.(:t p r o t e i n was de+,.e.rnfiJmd by the m e t h o d of Ludowieg and D o r f m a n (1960). -k ratio of ram acetyl group per 21,000--.1=.900 g of p r o t e i n was calculated from duplic, ate experiments. TA 'ram 1:
Nuclear magnetic rcsona~w(: ~')mctra of .N-for,~yl-31el:.'Is)~ aml the blocked dipvytidc N-l:ormyl-Met-Asp {1) ~- Va hl/: J ' a r t m'.rt
8"12 -I -(i 4-5 2"60 2-10 2"05
,%ingl~'t [.(u:k [)cat" p a t t ('z'n B r u i t iph.} I),,ubh't T r i pier. Singlet (h,n,plex
--
---
2.92
1";27
] H o c k e d (li 1. t'l,ti(l,: (I I ) 8-X'alm, Patter,,
.:\ssignmcftt
,t I, ,.
. . . . . . . . . -l-G !a~('l¢ b e a t l m l t e r n 4-.', 31 u h.i ph-l. " 2"1t".2" I.h ml*b:t 2.{it ) T r i plot 2-I0 Sin~lel 2-1)5 (.'-,m, p h ' x 2-0 [ SiJ'tglot . . . . . .
d
. f r.,, L
3htltiplet
iu:Imri~5 .
.
.
I
l Cl[.e
}
I
{
Ii t
0
H b
I
It
l-[ () c
!
tI
f
('IL_ 9
CI{= d
(I) 1 - [ - - C 2 - - . N - - ( J - - - C - - - N - - C ~ - ( ' O O I
{ ' ( ) ( ) | [ I:
1
I
C'H= .q
-
('1l._ ,~
C0()11 b
I, (1[)
[ !,
l,
("lla---(.{
I}
i
t.'l'l._, d -
I
...... N . . . . ( 7 . - - - ( ' - - - . N - .
i
:
1,
g
!:
A'.--G(H
)II L
i
]t c
c
F i r s t - o r d e r c h e m i c a l shift.-, a r e g i v e n .'t-; 3 - v a l n e s . Lakintz 5 == 2-10 fi,r t h e - - . S - . - . ( ' l l ~ p r o t , m , . r e l a t i v e i n t c n s t ~ , i e s a n d t h e . i n d i c a d e d t a ~ l t i p l i c i t i e s c.ozttirm the. a s e i g m n ( . n t s .
4.
"Yh,"
lSiseussion
For a n u m b e r of years the N-termirum of ~-crystallin remained an mtsolvec[ probiem. Alt.hough D N P - g l u t a m i c aehi was found w i t k Sanger's FlSNl-~-nmthod, t.he a m o u n t .of N - t e r m i n a l g h t t a m i c acid ~H)peared to be too small to be. sigxfifieant, in view of our knowledge of-the s u b s t r u c t u r e of y.-crystallin. ]3life, trait i n t e r p r e t a t i o n s of the discrepancy between the q u a l t t i t y of N-rorminal a m i n o acid a w l t.}m molecular weight of the sltbltnit-s were pos~sible: (a) The architectm'c of ~-cry.stalli~t cotfld be such t h a t tile N-termin'al a m i a o acid was a t least p a r t l y inaccessible. (b) ~f glutsamic acid was t h e N--terminal a m i n o acid the f o r m a t i o n of pyrr61id-2one-5-carboxylie acid nfight h a v e t a k e n place, t~ing closure, of this t y p e has been reported, e.g. b y Press, tSlggot and P o r t e r (1966). (c) C a r b o h y d r a t e could mask t h e end group.
N-TFIP,.M:INUS OF ~ - C I ~ Y S T A L L I N
299
(d) A c v h t t i o n ~:rf" t h e ~-Ln~ino e n d g r o u p c o u l d l ) r e v e n t c o u p l i n g w i t h li'DN]3. T h e l a t t e r p o s s i b i l i t y w a s l?OStttla.tcd b y ] 3 ] o e m c n d a t c t al. (1965). ( A d a) A s r e p o r t e d earlim" b y B l o e m e n d a l et, al. (1966), n o i n c r e a s e o f N - t e r m i n a l g h t t a . m i e a e M w a s o b s e r v e d w h e n t h e c o u p l i D g react, i o n wit;h F D N B w a s p e r f o r m e d in t l m p r e s e n c e o1" 7 .~t u r e a . T h c ' r e f o r e , p o s s i b i l i t y (a) c o u l d b e e x c l u d e d . (A~t b) A m t m b e r o f e x p e r i m e n t s w e r e p e r f o r m e d u u d e r co/tditio~ts t h a t r e s u l t e d in o p e n i n g o f t h e p3n'rolidonc: r i n g ; h o w e v e r , a h i g h e r a m o u n t o f N - t e r m i n a l g h m r m i e aci(:l c o u b t n o t I)e. d e t e e t e c l . ( A d ~,) ( ' a r l , o h y d r a t e negative.
gmmps couM
be r u l e d
o u t as t h e a n t h r o n e
reaction
was
(A,I d) As m e n t i o m : d Crarlier (/31ocrmmulal eL a.]. 1966), t i m p r e s e n c e o f ,~Lf o r m y l o r a,n a(,~.t yl g , ' - u p w a s v e r y ] i k e l y . h t t i m r e m t l t s s e c t i o n wc h a v e n o w d e m o n s t . r a t e d ~'it.h o ' , ' e r w h e h ~ d n g ex'i~lence t h a t t.he b l o e k i ~ g gq'oup is a c e t y l wl~ile t h e N - t e r m i n a l a m i i m a c i d is ~,wtl,~mi~m. T i m s e c o n d a m i n o a c i d a t t h e t e r m i n u s w a s f o u n d t o be a s p a r t i c acid. Fm'th,n" i u v e s t i g a t i m t s a r e i n progq'ess in o r d e r t o a n a l y z e t h e s m a l l a m o m l t o f or.her b l o c k e d l ml)ti
.
'
•
A CK X 0 W L E ]3 0 M E N'1:8 \V;, s h o v l d like t,5 thau.k Profe.~sor J. \V. lg. C o e n e n an<[. Mr M. v a n O o r k o m fro:. t h e porl'~n'manc:i:' of N3iI{ specg'roscopy a nd in t;erpregal~ion of tJte results. T h a n k s are d u e also t o Miss .J,do: v a n Tol, Mr' A. K o p p e r s a n d Mr J. R,e i j n e n for t h e i r skillecl t e c h n i c a l a s s i s t a n c e . P,.E:F151-~ E N C E S
yon .'\rx. E. :rod Ncher. ]~.. (1963). J. Chro~nalog. 1 2 , 329. ]3haeea, N..%.'.. john.,son, L. :F. and S.hooler3". 3. N.'(1962). Varian £'pectra C'atalogue, spectr~ 39 resln-.ctively 42,1..N~ttional Press. U.S.A. t31ocmendal. H. (1967). Thesis Univcrsit.y of A m s t e r d a m . .13loemendal; }I.. Bont., \V. S., ]3enedetti. ]g. I,. a n d \Vis.6c, .1. I-[. (1965). ]'2xTtl Eye Res. 4, 319. T~loemend~d. 1{. a n d ten (;ate, O. (1958)..Vature 1 8 1 , 340. ]31ocrnendM, 14.. Bo~3t, "~\:. S., J o n k i n d . J. F. mid %Zisse, J . I - L (1962). E~pll Eye _Re~. 1, 300. J31oemendal. 1;I., ]foenders, H. J., 8ehocnmakm~, . J . G . G . , Zwee~, _-k.a n d 0erding, J. J. T. (1966). l~roc. Syrup. d~io,'hem, l'~!/e (~_I'utzing, Gcrmal~y) (in press): van l ) a m . A. 1v. a.nd ten Care, G. (1966). ]3iochim. Biol)hy.s. Act(t. 121, 183. Firfarova. 1-2. I v. (1958). Biol:himiya 2 3 , 129. Gra,s,smann, '~V. and ~Viinsch, E. (1.95S). Chem. Bet. 91, 449. Greenslein. J. P. and ~Vinit.z. 3[. (i961). Chemistrfi of the A m i n o AcicL~, p. 942, 1016. J o h n Wiley &. Sons, New k'ork ~md London. ]:{ahnel, R . (1961). M rch. OphlhalmgL 163, 283. ]:[oenders, }[. J. and J~loo.nmnda.l, J:{. (1967). ]3iochDn. ]31ophys. Act(~ 147, 183. Ludowieg, ,J. a n d ])orfman, A. (19{J0). J3iochDn. Bioph!/s. A cla 38, 212. Moil'at., E. 19. and Lytle, P,. I. (1959). M nalyt. Chem. 3 1 , 9 2 0 .
300
H. J. t [ O F , N D E R , ~ E T AI,
])re~,;. E. M., l?it