Purification and composition studies of βs-crystallin 
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Publisher
Date
1966-10
Description
One of the low molecular weight proteins of bovine lens extract, designated as βs-crystallin, was purified by gel-filtration on Sephadex G-75 and chromatography on DEAE-Sephadex.
The isolated protein appeared to be homogeneous as judged by gel-electrophoresis and ultracentrifugal analyses.
The molar extinction coefficient at 278 mμ is 5·28 × 104.
The N-terminal amino acid did not react with 2,4-dinitrofluorobenzene.
There is evidence that the carboxyl-terminal group is glutamic acid.
Amino acid analysis was performed with a recovery of 100·9% of the dry weight of the protein.
The molecular weight, calculated from the amino acid composition is 28,402 and is consistent with the value determined by the approach-to-equilibrium method.
The protein contains 246 amino acid residues with the following composition: Asp22 Thr7 Ser15 Glu31 Pro9 Gly20 Ala14 Val12 Met8 Heu10 Leu14 Tyr16 Phe14 Lys12 His10 Arg16 Try5 (Cys-SH)5 (Cys-SS-Cys)2 (-CONH2)25.
The isolated protein appeared to be homogeneous as judged by gel-electrophoresis and ultracentrifugal analyses.
The molar extinction coefficient at 278 mμ is 5·28 × 104.
The N-terminal amino acid did not react with 2,4-dinitrofluorobenzene.
There is evidence that the carboxyl-terminal group is glutamic acid.
Amino acid analysis was performed with a recovery of 100·9% of the dry weight of the protein.
The molecular weight, calculated from the amino acid composition is 28,402 and is consistent with the value determined by the approach-to-equilibrium method.
The protein contains 246 amino acid residues with the following composition: Asp22 Thr7 Ser15 Glu31 Pro9 Gly20 Ala14 Val12 Met8 Heu10 Leu14 Tyr16 Phe14 Lys12 His10 Arg16 Try5 (Cys-SH)5 (Cys-SS-Cys)2 (-CONH2)25.
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Type
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Identifier
Experimental Eye Research 5(4), 255-266 (1966)
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E.+ptl l:;!/e I~es, (19116) 5, 255--.266
Purification and Composit ion Studies of l~,-Crystallin~
A. F. van .l)at,1
The Laboratory for Medical A~mtomy ,u+~+d Embryoh~ly, Stale Uttft 'crsity o f l :trt'('ltt, 7'he Nelherla.nd.s
( IIc(..,eived 11 May 1966)
~),:e of the tow mtdet:ular wsdghl prut(,i)m of bovine, lens t,xtraot+ (lt,slgnal4.~ t~s ]J,..ery,tallhi, was purifie¢t by gel-liltT'tl%itm on t-;epttr~(h:x (:-76 alut chnmmtography oft. ])EA E,,%.'ephndcx.
The isolated proi<,in al)l)~mn+d to li(, |lolilogoneotts as jtidgtut by gtq.vlt.t:tro|>)tortmi,*~ ailtJ t,ltrae+~ut.rifugtzi an[Hyscs.
The nlolnr exq,inetion eoefti<~ient, at 278 mtt is 5.2H x IOL q'ha." ~ ' . *e . r r l l i na | t tn lb~o aefd d id ilOL react w i th 2.4-dh~itrotltiotx~bi.tz~+te. TIL¢-rt; is ovi(lt:tt('(~ t.ltat tit(: earb+Jxyl-te.rmbtal group is gluLtlrnie acid. Atnino acid t~,liid3,sis +vau perfbrmed with a. reoovory o f I00,II+.% ,)f t ht.~ dry we ight t ip l-hi+
[)Tt 9 t (,+jltl, '.I'tte rnolet:utar wt4ghL, etdeuJatetJ from the ftDlitio tlt+id corn+misitio+t im 28,402 artd i,
voltsisleid~ wilh the +rll[I.(['. tlc.-~':rmine4 l>y t, he approae|l-txi-oqtfiHbrium me.tluid. 'l'}z+~ proleili ¢ont,o.Ji~ 24(1 I+n)irit) acid +~.,sidu(m with the fol iowit~ (wmU~.mitiofl: Aapt l
Thr: St'i+ul (i|ilil i Pro~ (.lly~o Alait Vit l l i ~ill<-t.~ llcli io ][x, l i i i Ty r t , Plii.%t l ,y~i i ltisio h rg l i Try s (C.y,s- Stt): (Cys-- ,%'4 - (-"YSlz ( - -CONt l i )~-
]. ;nlroductlon
It, his ct'lssical work Mfrn~r (1894) dcn~olmLrated, on a pr<qmr~¢ive basic, thai.. (.lie. lens was composed, of three soluble proteins, ~.- fl- and ~,-cr)~ta||itrs.
l,+~ter on, fro'c-and ptq)t~r-elet;tr6l)hor(mis of the soluble proteins of Lhe vv, rtebrat:o let:s supported l;his classification (lies,~clvik, 1939; Frattgois, Rahaey artd Wien)e, | 95,t). Chromatography tm ion-exohaiigers ( Papaconstantinou, Remfik and Saito, 1962 Schmidl, and t(m Care, 1961, 1962; Specter, 1960), inmumoe]ecLrot)horesis (Manski, Aucrl)ach and f {all)el"c, 1960; Manski, I lalbert anti Auerbaeh, 19t31 ; ltran~ois, [Oabaey, Wiemu and Kaminsky, 1956) arid especially ZOla~ elecLrophoresis in agar gels (Frrm~ois and.Rabaey, 19.57, t959; Rabaey, 1959) revealed the het~rogeneiLy of each of those. fractions. By meaJm of micro-agar eleetrophorcsis Rabaey (1959) fourtd 11 fractiotm itt extracts of cat~le lenses.
In particular, i~he medium fractions were highly qomplex and 'showe~l a tmaeatred band in t.he pherograms:, in which complexly r~olved fractions could not bt~ demon- strated. However, tim slowest part of caruso medial fr~ctions, called Mt by" Rabaey (1959), tbrm~ a sharp band. Since the pro'~eiim with ittCermediat~ mobilitiea in ft~¢ and' zone-electrophore.sis have i tsually be;e~ hldicat~l as the fl-crystallirrs, we prot~ ~o call this e_~mponent fl,-crystalljn.
In this commurtication the isolation and some propel~ies of this protein will be report~q.
2. Material and Methods q'lie, extract~s were prepared f rom bov ine lenses as fol lows: i tmnud ia~ly af*~r s laughter
the eyes were removed and t rans fer red to the laboratory unt~r cool ing, Withit~ 2 -3 hr a f te r
This work was supported in part by grattlm fro'in the I)uteh Organization for Pure $~imatitlc ICtmm, reh (Z.W.O.).
255
~edl ..%. 1". VAN 1~,~,~I.
t.hu dv;.tt.h +.ff the tt~jim+d~ |he. ]+:n.-~,~ w~..re t+xlq~ed, w+LMt~:d wit.h cold phy~iu . l ,~ ieM mdi~v,, d eeapmdM.,~(l, su~peuded in ,;o!d w,h : r (,3 ml /g le..u,z) ~ml ~t i r r , ,d a t . .3 :~) rev /udn for !*~ l i t ,
Tho e, ort.ic+tl i~×t.rttt.t, t)bt~tinetl ill thi.~ W£ey ~s'a~..t e~.wtt.rifuged ~tt 7;3,~XJO .~ g ill a t lec lcn l , l~- ,qph~t-o l,, I ecdLril 'ugta fi.)r 3t.I mi l l t.o re tnt ivc t.ht: inst;htble, tdlJtttil¢~id, 'i'ht~ op:tlt,.~c~onl/ ,upern l l t l tn t wiu~ lyoph i l i ted attd ~Lor~d ttt. . . . . . 2 ( 1~ {~3 "
No ~iilTer~,nc.e, itl (:Iivotll~tl.ogi'itll~Ly. ~lgttl'+- atl+l ii~lttittlxo6|e~:f, ro ldtore~i ' t wet , ~. ~,b:~erv+,tl b+++l.wcotl I,hi~ lyop| f i l i z t .d a~i+l froMi m~tt~:ri~d.
Tlt,~ txt.~v l l t , ,V f i~rm of Sel?htuh,.× (I-7,5 (i-+harrta~t(Jill-Ate. UpI~/~i~h.t, Sweden) wa , u~e.d, wh ich g ivea +~ bt':(i.or r~ ~otut,/utx ~lxtl n h i~h,w elut io+l ~'~tt:t~ tlttt~ l+|to, fo rm we uaed pt-t:vio~.~ty. q'he gv~lilu+ ~yerv ~w,dh, lx ih O'0t .~[ 'rr i~ bufr¢.,r, p l I ?gJ l',~ "~ hr. '[ 'he fil~t~t gel p~rticle.:+ w+~r't~. x'e.m<,vlrd by ~l~~t'.~', t'~t~tioll atxd t.|d.~ procedure was r(+.pe~tLt;tr| fi-li t.imt'++. A rt+,r t,h+~ tinafl ttetr~t~L~- l.io~ the t.hit;k P+lush wa.a .,i~apenttc,,d itx ~hc eh.lthl.K buffe.r ~.~l+d dettert~t+t.+,,.i fur IP; nxiu. 'Vh,+.
l,~ttt'it~g t.~>~+~k 'pht0t~ tit, roont teltqu+vttture, ' l 'ho gel bed w+.as w+~,+hed ovex'~6~ht, wii ,h l~ufl',.<r ie+ the c - ]d r+~ottt (.I<'C) t.,, let, {t. ++t~d,ilizt,. The col+.lltitts ilse<l h++d th+_', dim,.~n~io,,~ t,t" ~.5 +:m +.+ l(~.) <~tix ~tit(I ,-,r ,I-,~:; c.tli :< i(~0 ~,~.ilt,. The . +ttnotmi.n of .+ample +tppJied were 1-2 ,t~tl 8-, <) g, re,~q,~+'ti v,: ly.
Ats elutillt~, butl't;l~s were t~s~| lt' l M '| 'r is I|(.'.1 i+tt p J l 7"3 |'or Lh,, tirol, ,..~"+t tilt;rat,i,~lt "~lul +~t
l,ri'l++lgbl. (xl p l l 7"8 (c,r p l t b.t3) wi/.h nmn'i++tti0.. Cllrt+,m~tol4r+tpbie :.,h+eld~ ~vl+re. i+t~+ttitt+t'<,,| by ttlt ul(.t'ttvi<de| del.,.'rt'tov tlllil ((l~,'i~-:t'~t't.] LI( I ' : . IL+ppmala, Swedetl)t:,+nt+e<:te+l w i th t.t vllopll+-I lu, r r t .etwder titl+l It'd hit.t~ u t'raet.i<m etdh.+etor ( I ,K IL ,'gwt~t|+,l;. t.~r t{+'+,+li.+ ,qwit.m,.rhtltd). l f ,) lh frt~(:l.iort ¢-oll+:('tt)r,s (q)++r~t.<,d (.,n +t t im, b,t:+i~.
[) I t ,rl~t+etrophotx~i~+e.t.l;r. <}'ht, J'rnt..Li<>lls were. c<+lI+,t.+t,e.d ill t,lto. +tl~prol)ri+;tt.+. lllltltllf+.i ', ~.tlpir vt)Iti++il,:+.+ det-e.r(tsir~i'<l+ +it+d l.h<..v xve.t'<s +Ii+I13"..+<~¢I ,'iti+l ]y++ph.ilizt+d. Aili¢,~+-<;xl:htt;tb(e (+itrotl)+tt<+- i.(l'ilpll, V ~,~,'l~ [>.+~+'ri~+~l +.,ut <;n .[)f'3Al~-Sel+lmdu, x .,%...~+O m<.+diui~i ~r;id('. (i,+t. +tt+,lbl,r +:i3[L ei~ p:tt'it.y 3- 5 .:L-1)- .'3 mt+.<lUiV./fl, l'tivt.icle, sizo I<~J--?,7t+ m0td+., t"Jtttrllittcitt +/II, t Jpt,:mhJ, S~v ,+l,m). Tilt. + i-lillt.~i'iFII W+I'+ Swotleit ft,r 2 d+tys ill t'.lt+" st~lrCilil4 hUffl'x' and tt io fille..+it. parl>ielt;s re l l i t lw, ti iLv itef.'li+itnbi<,i]. AJPl:cr swtdli.,tg, t..ho I)l,.]+,tl~-B~++tihad~+x wlt;~ ,,vJl++ht, ll t:tJll,+-te.l;uli<.'elv w i th
• 8.,+ wiiAi l i-~ ,sl N~t l . l lP i J i l'+. I N t lC I , wat,++r, i),. l +,+ N i i l t l l _ +iii it ~,+'ill,¢rr. ll.. w~iJ+ hPl+li l l | l t~ t.o p i [ "" '~ +ill{f wfiv.lied w{i.+h the st.lll'tiillg bitff~r.
' | ' l i t , eOJtltilliS {tiilFion~il)llV.: 7"~;-..2lOt.+lll)"livel'¢, ir)<iliirt+.tt..litid sefli.tiieiit, t:+l l iy grl<.vlty ,,it. t'fltTtiSi i=t+llil'ie..llit+lll'f++. <]['tilt le . l bed w+t.~ st. l ibi l ized hs" W£t+t].ll l l~ .i1+. ovt.+rliighl; itt.-I'~C wit.h flit, P~t.nrt,itig bllf]'or+ Thc'. ml.ilqlle.'L V+i l ' y i l ig I-i£q.Wf~.P+ti 700 i t lXd +e.7(7)II1~ i l l i~+'.l)>trltL~ +.~.xtt~riirxt:-rti,~+ w !re di . ' l .~olved ill l t l ld lti~llv.'+t:<! tt l i t i l tst, t;tt~2 ~t l t r t l t t 7 httlTtr.i +"
The olilt.l~Jil ts'lt~ eff~,eli,d b'r it di/~COlltitll+loii~t ~rti<il~;tit. (i it i<tni+: .~Lr(:iij_~.l.h ul/<l p.! l ) "rh,.: t ' tdlowilt l t ['ihoi~trliilt.e tlttlGi++:r++< were eltil>JOS+<l ( l ) O-lIO,~)M id l i~-~; (~) 6"f)l ,~t 1,tl. 7. I:
(3) (l+(.tl. M p l l 6-8; (.l) t)-f.l~ xt p f f i3-8; (5) O.(l~l .~! p i [ (l+~4 t-(i..l .~I Ntif~J. :+% (i-I lit.~,i I , ] t )TA ,~oiut ion. brc>ught tcl pl i 7-3 w ld i Pl l l it l l l l i l ia Wits lid~iu'd t<~ I;ll tliilrtt-'+i>~ --rtL,.+ frat..t.ic+ll.~ tilt<.lei" l,hO differecil. IW, ak+ wt-re pooh, it , ~lii+ly+ed uil+t t.vophiti~,e<l. Asz:,r-
t~it-cii'tq>iiort,~ds ~,,,'~it~ ipi~rfort¢l~:d ll(:v.<~i'.iilig t+.+ l..|ie Iiri>oodtli'i, of lili'it.li;1 " (I,t.).rtt. ~'') oil ,<,!7,t+'-~ ~i" }7 < ' I l l - : - f t i l l . Th<~ r,.,t~li.i~',, lilobititi+'.++ wt:re e.alcu}at+>d wi th t.t~+.,+ li~iel-hot.t Of ~IVit?ll)l~ (l~,IrSt)).
\"+.'.rl.i<.~+il si.::ln?+h gel +,let~IrtJtlhor,'~sh~ w+l~ earri+ed ~,+~t +l:.~ d,:,.~+erib~,tt by Stilittii<~>+ (l~,t.">+,i). "['|it+ 71+1 I~ufl't.w xt'~l_~ O-(i(.+ .~1 ' l 'rbg t.iLi'ltted wlt.t+l h~icio. +reid t.<l pl-I 7.t; i i i i l | (i,,~t M t>oril, :lCid: t.itt'.<tl+-,t +<, p t l 7.+I w i th Na(. J l | , wll~ +.,mld,Lved (,t:" t,ridt4,+ l ltiffer. /4]ie+:f r<lllli,~r,%~i.+~ i.,~,+k" pl+j<'+.~ +it. +1""{7 tl,,411:i|l.'f f~r ii+~ • t~ hr .
3 ¢., ii~ a I.r|t'+'+'~t'.+'t+l ri|'tU.~+LI +~t~l++l~,'m'S ~,v+.,re. Illtld," i+t " +: " t ' fLyw6 ;lir-<|r{t'+,ti ti lt t'+tt'Plill'iftil{<, 'l'tl,: [tr< t+~i+~ wll~ ~ti~:~.,tv,~l hi h,,~ri.liLttr;~te tllift'+,.r ++J+ i~|I 7-11 ~ntl ~dju.~l.~,~i w i th N:.tl' l li> al l i,.mit- .~tlei:ll~l.tt t , f 0,T i i / t .
[ b,t ~, i .no] i ,v ! . l l i l l + %,+'t.il~!tt, vi'::i+~ ,b+t, , r t lx i r tn , t tti,, ftT~+;~ i+llqtr<'>a<'ti+t.l~~~.<'~|Tii l l ' l lt+tli<~il-i 'qil i! i l .tri~tP+ . l lt~>lti 1+i, dt° l , . i ' . l ' t l l{ l l l t i{+it l : 4 l i t +~it'lrt'l'f'llt ro|.+)r P+p+~_i:,f].~ xvt~rf >. iitl"ol'l14~+,~+l,~l i l l t<l ~t Ti'~+tlt lq.<ii i t:{,,t {'[+t-~ttli.tll i ifl+ i ti~'.~t+i)+
I ' L . ' I t l I , ' I I * .A ' ! ' I ( JN ANI ) +~t)MI ) t J s l ' l ' t l )N ~ ' l ' l ' l ) l ) , ; , '+ t+l ' IL ( ' t IY .~ ' t 'A I , t - t , '~ ,;;~',7
Ami lu , +u:.id-itti+d)',~+'+ wi,r(. > t,(.~t"f+,r'iti:->+l w i th )t 'l',,t.hilh:<+s.i i l . t l l l ix( ' t~l"llt ;-;l~.t*.t,) I.+"l i it-i +et'lttlt+, ~ I.~) t.)lt' iillHi.lll')tl ' I l l t'i~,;,. , l l id ,Mt,l't.~ (IW(iII). T, , il~+i,'h] l ' l t , J~;itt~. ~ )#i r i l l , ' I.iil'+t".>t.r'¢ |ti~J~'. ~" :~ 1 i'tJl'>ii~liftt.+~ liuff+.,r p I I E,..~) was ll~'.>tl iil.'(I.t'iti| Of i ~.1 #,<iltli l l l i lt+'l'i';il<; ),iit,l'i,r i , I t r,.D (M,~.,i#~J,-i~_ [t¢.~!'.~4<)1<t~1] i~ l i l l l l l i l . i t l i l ' l l l , i i ) l i ) , tl 'ttr,+l. ' hydrv ly~i~ tixl~,.,.,~ w,..rv t~.~¢d, vl~.. '7.t, 1<~ +,)i~l ?:~. l,P
Tif+.V.llt.olJttitfi lt"lt#i t l l>l.e. l ' l / l i i i i ' l t ],..V I. lte l)l,~l.h(l<! (if Spi ,>~ +)l)il {'litl i i ltt. l '~'; tl'~+.l~q ,~,1 h~: ttl~, s l i fq ; t i ' i i l iho l i l ' l i l i~ l .F iC t i l i , l .h , id t;'f |J+>()tl'*l¢ili si l i*] /~tlf..,rtoli ( i l L | l ) ) .
'J'}lt~ t~'iti.l~r-(-oixl.i)l l l.~ <if i.[l(f, ti~,<,i.elll r i , ; lut it . l f t~ wt~r+2 +b-,t.~.llt~li),,,.f l )v t t r )o i l t~ I.*..* v-lili~l+-l~t~t weight, ~t l(.)f)"(!.
[! lt i '~tvitdt~t; ~+lit;(:tr:~l )~tiiil}'~,ii+ wi,:+ It llil+." iiX" l it l ' . ' i l l . ~, t,f it tl;~w~'rtl~il~ I ) l : ,~t,.,.'-l+',qt)t,:t(,. I l l i ' i .( ' / ' .
• o
~- l . ( ) f t l l l J l i t l ~t/ . ) l l t ) ditl4~.ltl l i l it i|. i+~ill~ V¢('l'+' l l i l l i l i : ~.%'ilt~. ",~-,:!l ltlliilf~.,t!fit~,i~.,1,,,lil.+,t*.+~ Jit.ct~tll i l lM. i t , lh+; l i i+~i| l l l i J <~f l ,e* , 'v l~,.i .(b,m'rib(:+l 1Lv | i ' t ' l i+! i i~+' l - ( ' l ) l i l l i t , l l l l . t ' t l~ li!l<t 1~ .~, %: i lit,#'.':lrt
(.~leil¢,~lt ' t ' l l l t t(r(+ll l i C]~'(~'/'lUIIUli.i+'¢i:'I tv,,r(, tt in. lb- wdh <'J l i l ) , ,~cy}~,. l l t i~|:t~, ) t - l i F l ~ (b,I ~ttl i l i l l t~r t~.tt~.-tl~lf+() SiI~i+lt+ (] Jt i>i ' i l lv lt l ( ' , i i l l t , , i t i ) ' , S t . J,,,~il~, 5 i , , ) : l - , | , , . ,+'ttl>,.,t hv |l+lt-ri.+ i i ; r ) l i ' l i k , ,1 .{J i ) i l l ' l t l t:L .tll, It t.i.r),r)) tl.I)ll |).y .1.tl)' ti.vd rli2.tll+tl.?':4i.~ tit+ -l, ti, '<i <'(" -:% k i i l+ , , t i . { l l i i~l l tt-{i~l ~'fll'i~li'L ( I ~.).'l~ ") li.q" +ll,m'r' iht0d i.)J' <|++ I,;i l,t+l.xii, T , , r l f i l l i l l ) t{ , | l i t t - -~. { 19+.=-li.
[tj-vu s l i l l t t ty l i l :yt /2r't>uf+-~ wet(, ttcl+'tl l l 't l i+,tl I,V t i l r : l l i+)* , t ~'sl l l p-h),. Is, .x,, lt , :-t ,~li+l-+~,,),~., ° tlcc+)rttint~, lti l ' Ill+'1 I i , - t ~,f i+,.l.,. W l l i l , . i~ll!i All|"llt~t-li (1 li.')tt.I
.t I I t '~l lt- ,
(v~u~ t),Lm a.n~l.t~,l (!~lt.~;, ]~.)tiG)rt..w..)d,.,d t t,;Lt ~,.,.',rV.~,dl~L ~:c~lrr , .d ~l~ t I , ~ ta~l t~r~d,,t~
[*' i~ttr( ' . | (}.t) ~how~ iI / ) ' [~ i~, J ( ]~.~Lr i |~i t i on / . : i l rve ( I f 1+2 b[ ¢)f l~<:~'i~! J~~l.~ ~:4.~l't~.~ ~Xtr~! : f
wit h ")ll }ntemml dia.m(:t(~i" ~H" ,-t.P} t.!t"i+ ~'~'t~e h)ttd,~d w}. th8 g of t llt: ~.l~ti,g'.. +.~xtr~,"t.
w<~.rv i l fcuti l ld-t , : i .y r,,~ol~n:d. "J"jtts ~,'.h p~:.~k m~dl l lv ,ti.m,i>im;,r~:d wh,'~, t.h,; l~'~,~ ,,xl.r)~+.'t
ub+ct.r,) i ,horesis of th i s fr+~.t...ri~)i,, i~ fl~,:l, lil.s(~ t~otcd l~V i~ i i rk {i5,3.1.1 .~fter g,-i t)|l.tatl.h.,~
))}g,b),r than i,i. EF;O nV.~).
r~tri))" du)u)tt~d/~z, J]~ at~d ~,+er'5"sb~dliri, aud pt~:.a.,.(:ry.~A.,tllitt ( l:qat~.: ! ). I~x~ttliin:t(i()t~ (')f tit(: Jront. iltl'.) back purl. ,.:r |<,ak 4 ~how,.,~t tt~ltt o)~e ),.-c%°stia||il~
I.y+.)~ i l ie ~l-(;ry~_t<d|{t~, ttt~t[ i)t('.-~t-cry~t./~}.l[n +.-.m(.'.rgr~ iti ).ho fr,.m(, par(. at ;d tt~at ~.~t~r~<J ?,i-ery~t.;dli)~ c. lncrge ~ll t:.h+ +. i.qr(:.k |)+~t. cif the. | )~tk ,
i i(} ~. t)f f,.t=ez(.~-dt'ied Icn-~ oxl..t~+.-.( )'Jold,~..! 8 ¢.~f fra(,,th)it .i "tit al l . '|'tt<~ )~.)|~.(| grott~,s 4 ~v,~r,, r,.!fih~++.r¢>tl oi l ~el,hadt.,.x t . L75 . .An +*~)),)wn in |'-+'if+. i (b ) li~,~+, i+~)i).++ tma~; (fr+e.tio)i ~) cmerlf<:d ill. the mtm+s t~3,~iti.ot: +m it* tit,:, l i c i t 14,,t filtr,+!:Itn,..
~t.are.]t gel eh~ci.l~)i)l)or,s~is o f fr~tc~tio~) 7 r+~,~'c-aled ~i. I x~ i i t | it: l~!',~c).~+;ity {lq~Lts ~.t, A.~ ?tll.tt/,y O~i l.t lili/+lJ.! Ir.~'JttJ+'t [~e tfemc, n.+tritt,~.,.|; )~i 7-ctv.+t++)lht (h++id. i.-P,)< H,-'cC+'+t+xtti+:~
~,',.'~ .%.. I". V ,%.~ lt~%,%I
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t;'t~i" f lu - itli, r:irh>h~!.. / t i tsor l i t . i i t t i l i le; i , - i t lr l , l i tel it , - i s+',ll-t're<., l y t~ l , t l j ' l i ze t t pt'~t~,.il i was , t i~,,+,dv+:d i l l 4}-I)t),¢1 ,M ] J l i osph l l L f ; tmf f<! r ( t , l l ~4.2). T i l l , . l i l '¢ltt~ill (:t)lXl::i-lii.l 'nli, ili +|i'll.,.;. 0,9.tl:i.~> (d ry ~vtdTli l .] , ' i ' l ic; >~b~<Jrl)t,i,:m f t i l4 ' t : i . l ' i l l l i rev,~;th,d a i l i . f4x i i l i l : l l l ill. A~ l i l l tt , ! l i l ( l l i l i i l l l i - t i l l . t i l l ll.[, ~')(Ji lllt.z " ' l ' j l ( : fq>t.ic'uJ ~h,+lis]t+,y i " tLt i ( ; i i ] . t l . 7~f J i i , / . , i l j . I , . 21..~', ,(17, 1,rti,q. t'l.~llil~l l¢.l Iw~ "2" 17 ltii<.l I . lt[s %'lt[lit +, Sli~[.(tr:st.s Llirtl l ri.u<~l+,ie +ti:i, ts I¢l'l, ~tlt~(, i l l . . ' tV i l l i i.he. ;t l l l ~Jf <| ry x,,-t,ig, llt. ( l r>>l . ( ' l ' i i l i i l i t l i l i l l l ,+ i t i l I/'5 'z 5"{ V I I I I I< ' , l i t ~78 il/#X, t t f + [S ' i i I t l i t l ill.. 77(1 i i t tt i I l l " 1 .~'~'1%%'l'.1"t; t : . i i l t+ l l id i t * '< l_ I I ' [ l l '| 'h+" Il i i l l+ti" t ;xt i i l t ' . i it>i| t : l l ,~lth' i+'t l l , it++' i i i l . ~7~ i l l / t , e i l l+'t l l i t l . t ' l | ri.i>ili ih~, i i l o l t . ' c t i l l t r we i j~t i t ( , r 2~- . t l i7 . i,,1 ")-2;"1 .." |iS t .~i 1 t'zti I
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As, ,+ui l i i i :g t.|illi. +~ae.ll L.vro,.-,yl i',:si<lll¢, g ivvs t, , :<.Jni.r i l ,ut.hui , , f 1-3.1 ~ I l l a i~, K ~.ltd t . ry l~- l .opht i l i o r F~.FIE)× IO :/, wt:. t'.lit(Flilitt~,d ft %-tllllf.; tit' ,1.t32 ;~. l i f t .~l i ('.1!t .I t 'rttl i l l}ttJ 1.li<,+4iil~" +i!!ttl t r .V l l l .o l l l i l i t t i :Ol i l .o l / I t i r l t t l ; }+rld~..-itl, t (i l iD.({ f'J t'~"ai~ltit'S, res l i t : ,St iv , : iv . The. r i l l i<, /<~,:*i'. +if I . ( i7 i~ i i t l i lt ' , t'llli+~(<t (tt'tl~t, --- i ' 1 '3} l 'Otl l i t{ If'i," %li'etl i l~lt' i .r l i t t le7) f~Jr e~+-:t.'~l':ll
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i ' l l . i l l ' l " l t t ' l l t , l ' i l i S . Th is dl flm+mic.~.~ bt,i<ween i.h~: OXl~i_q ' i l t i t : t i l i l l i l l i i ] t t it" l .het ) i ' l~ l i l : f i l v i i ll~t {:~, n l,+, ,+X ldn i i led t>~" i l l / ~.+llhil.llti~.(.tl a t l s t , i l l{ i¢t l t {>fr the l l .rt) lt i l l [ ic, r#.,.4:+.tilt,.s tlS :1 r tmul f t~f l.[tl'.ii" t>l'l.'C'f+Ulll'++~- i l l t | l t~ l+r l .~[t , j i l .
[t.I¢~&te~,i,.lr o.'.~'i#]l~l dcl, 'rm i,~ll.7<m.s
ili.l.o ~l. "|'raul;l',tat~ plr~t., a.l'~ giv+~lf in [~'ii;~..t. As Be.e.ll {I~ tjl.i~ |ig{t:rt% t i,t~ '|~l;alltttxtt.rl p]ot. i~ l im~u, wh ich i~ charat:t,eri.~l ie. t'<,:" ~, h+mu~gmleot,~ ~tmc:i,,.~.
| " rmu lho sl~,}m of t im r~14r,'+'~'~icm 17m~, dmcrm[m.d t,~" rm..ans cq' tht: l,~,aa~, t~.qtt.<~l-,.,~ vlmt.hod, the z-t~s'+::ragc t , ,dm:od upimre+nt, lnt ,h,eu[ i t r wei.lcht, ;ll,,~,, t l.. ~p), t'uit by t'l~h.'u-. lat,w}.
+<ka+[gt~.it;g t~r,,," i.t.,i.t{iti+..ti,.~+, l imit, un<:l ~',+ith ~t-+uld+m:l de.vial i¢,ns e.+ilt.+ttl++tutl tvil.b gx+'+,'-2) d*~gr,.'+~.~.~ t>f l'ret.,|o**i..q, v<.dl,, :. o f (O-";I~:~-Ci-(K~} .* iD "l xv~rs l"+~ttitd ft,r t hi++ re+lm':a~tl ,m,iee.~i!i+tr we ighL
T im tmrt.i~t} +l,+:ciih: v<quilt+.: {+<') w+ts +:+ilc~tb,++,+t +,t+e,,nlin g t,.~ the nmth,:ul cA' ('<.*Ira told. Eda:d l ( 1'.)-13}' l't'+,ttt_ thc tt ram,.+ i~ch[ t,.++ml~osiLi<m, t+'or t hi, sluu-+i~ic x.~ltltlt+.: <if t:.ye:t ill,: t.hi.'. ¥ i ihm o f 51eSteok in +til<l ~l .+lmiit i l l ( I{ jhO) iv+i~, u.Sedo T im c. i~lcul l t l . io l i .v ield~'d ,l vt ih.u. l+l|" (}+7~7 l+ i t i t ~ {i+t;i~ T t l t j l i . ~ t [ ) , 1..)4311•it.%. + llt++l~,Sll+trtlil.t,.~ill.8 t~,t 3'a( >. U+i l i fg it. t (t l rnl Jl)'C:llot:~l+.+.Ler,
.i l ' ( ' l l l t t. hi,*t41'7' d l i l~ ;+ t : i lotOet l inr we. ig l i t +.~f 2SOt.r!i ..:+--l(tl~.i w+L,+ ~ttvi~. a '¢~lim'. of I'tRS2+, +" +;alc.ul~ited fro" ~J,-t, ) i t .a l l , i t .
l'I'l{II.'lt'.A'l'lqlN ANI= ¢ , : t Jh t l '~)H l ' l ' l ¢~N ~; ' l ' ( ; l ) I I . ;~ i~I: ~, ~'ht'V,~'l'Al.l. lX ~I,,l
A ~,~ ira> a('id ~:(',m I~'it i<,n 'l',tbh~: I. rm."x~r¢l.~ tim mnim);~<,i~l eOmlU)~iticm. ('~.mven:i~vmlty, tim v,tim, s f¢,r tt, r¢:o..
n in,:, se.ri n,* nntl ;m~id,:.-N | 1 ~ w¢,n., ex tl.t'uI)l)ll| 1, ~(! t (1 ~r~'.l'l} {i|'~ |.(~ t¥t" hyd r~.,lyui.~. F(~r v~lmr, ~tll~l i~,.detl~il~(! t.Jm ma.xbtml vtdue:~ of 7"2 hr h)'drolysi.~ tiara, w~,r~, t.~ken ~m,l f,~r t1,~,
I)upli(.ati:e r~m, lyse.s r,.'vt:uI an ~v¢~r~tg,. meu~ duvi~tio~, ,,f l'*.i.. 'l'h~, tryl,t,..,phuv~ ,:o~lfetl l , ([,~ve.rluitm,| n.~:t:~.~r,]il~,g t,) ,'.q,.l,i,~.~ ~ ~,ut ( :ham|mr~ (t9 i9}, ~tgr-,...u.w.,.ry wol] ~'i~ h
3.38 w~:iRtii. ~.'-i~, z'e-~l"<:i~ivel.g, t~,,:uus,' the t'(.~r~m n~'tb , - I i,~ v,-r,, re.li,d)h~. ~v~, ~',tteu-
Va~,I|I(',S, 111',' g.~'l',l { i l l ) " ,'X|.r~i})~]lt, tq:~[ (Ill{| l.-jl{ =- l l ta lXi l l l l i l i t X'IIIID.'.'4 t't.'tHtt l | | , , {.ltl"~*~" l~w|r<,lv,,b*
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t]me~. T im :era.~vm'v of I0~.).9'].i~ ,.>i~ u weiu~1, b~i,~ provid~,~ :~ ch~r:k (m qhe ~e.l i~|hy ~d' t h( ~. ve~n|t.~ :~n¢| in ~.ddi~i~m il ('.~u be co~e]j~,h,d theft, th,, pr~.*~-~~:,~ ~f ami~a, ~ '~[~
mob,cuber weigh( (rom the Arehib~dd me~¢~r~:n~,rc;~.,.
i~.d,-g~al l}l,~It~])~',~'* are given.
_n , . A . 1.'. VAN I),%,%I
'['h(: l>)'(,tein (:~mtains 2,1(5 residues. Since l,he, differc~u.e b(:twe(,)t (u.sp~r*.ic+glut- t)mic a(fid-.'tmitle) and l.y,.sine+argirf iac+hist. it l in(Q is 12 l:,t,,s'ic g)'OUlm, one might expe.ct, ,~t slight, ly alkalim., iaoionic poiut,. This expla ins t, hc slight.ly cathod ic movemet~t. of t.he prot.ei,~ ill the, starch gel e[cctrot)horesis a.L l)].[ 7.6.
¢ • I ~ l .h. t, it.raLion w[t;h /~-hyd.roxymcrcttr ibenzoatc rcveah;(t 5 S l l -g roups I)(,r )nolo iu (.he ~tad;iw: protein tat, pl:[ 7"0, A s l ight ly h igher w~tue (5.5) was obl;a.hacd whe~ (:he twot(dt~ was <.iet~att~ra~ed wi(,].t 5 ,,a gtt~,nidin(;+.[[(31 bt:fot'c ~reaLment witch p -hydroxy- mcr(:.urib(;nzo~t(~, a~l(l Lit(; opt ical (tetlsiLy was n~casured af ter 7[) hr. Thu.s it alq:)cars that, (;he I)rot(:in posse, sses 5 free su lphydry l gro~)l)s ~t~lti p robab ly '2 cyst.!he resi<l~(;s, I lowo.ver, this has ttof, as yet; beeu e..~tablishcd conclusive.ly.
TALH, t,: [.
A ~, in o acid compositim~, of ,B.~-cr.ystalli,g
Ami) )o ~to, i(I res idue
N(:al'eS(, hnLegr-~ I
Aufitt,) a(; id Agmm~t~([ ra). :ktnitl~) t~cid number ,d re.sit lues/ Mit~im)tl o f ros idue~ L :n lcu latcd resi(htc.s/ :~mi;~o ac id ]00 g O1' lll.O[Ct~'Llhtr {'(w 2,~'0110 g Ittolo(~ular 28.402 g o f too' 28.402 g pro te in a we ight ~' o f 1)rotcin wcighl , p r , ) te in (>f T)rot.e.i*x
Asp~tr t i c a~(:i<~, 8 -9 J 1292 '22 2S424 21 ,1)~ "22 Thrco~Ji n(.* 2 .b2 c ,10 t :2 7 2808.1 7 4 )8 7 Ser i n6 ~ 4.68 (" 186 1 ! ;3 27.(~ ] 5 ] 5.2ti 1 ;5 (}lt:'bnrnic ac id 13"S~J {)30 30 27900 30.;54 "~1 Pr olin(; 3.22 /tO 1 (l i) 2714.1 !J-42 ~) ( . ; lye!no :?,-96 144 1 211 2g,q2[} 1!)-71 20 i~. h~n i tw. 3-45 20fit'l ! -I 28S4 f~ | .'|'7!) t 4 Vtl l i ~t~ 4.07 d g-t 36 12 o(~,1_3,. ")" ") ! 1 436 1:2 514;tla i(,n i nu- 3 .ti3 3(5 t 4 .~ 28912 7-,'-¢(¢ B [s<)le,~ci)~(; 3.95 d 2~(;5 10 28(~50 !).!) | I 1) {.~.u(:it~e 5-54 '?051 ! 4 287 |,l 1:1.~:3 I -I ' ' ' _81 , ) _ 16 -1 ° !(~ 1 .')'r~.,slnt~ ~,t-2(i L71;2 111 ,, ' t -) - | )| Wily J tl.[tt |Xi t lt~ 7":~0 ".2)044 1 -I 281 i I (; 1 ;{-c.){) 14 l .y~i lv: ,)-17 23.t3 1 ~2 28 11 (; 12" 12 12 [ i i s t i~ tiu (, , i -711 °811: t 1 o '1811' ]0 9-( . )2 l ( i Arg in iav ~.)..~19 151;:[ 1S 2813-1 | S. 17 1 ,'4 A=ni,.h:: at~n)t,ni:t (1.39) er 115f (24) 27672.1 2.1.1i8 1251 "l'ot.al h ) t l f .ey , t i ne 3.2 ! i 3 178 .q 281)(.):2 S-94 9 'Fry pLopl tat t ~ 3. :20 f 58 t. 9 5 290.1171 4 '88 ,5
'1"< ,ta ]: 100"9-1 28402 ~ 24,6 28:167 h
5]~,~tt~ res idue wr ight . 116
r~.-;idue n~, lecub~r weight ) / . It) l) )) Cttlouh~Lod fr()m tLe rela(. iofta|f ip, of t lm!no ae idres idue it~ protcb l "
~" Value. exbrap<)httt:d, to zt:r(, | . imu f)f hydr(dysi .~. d Vi~Iuc o f the 72-hr hydxo lys t~e. t~ A va lue o f 3"38 {weight. l)ur t~()tat,) was ol)tMrtc, d by Oa(, Sl)ectropht)t , omotr ie ,nethod ~,f ( ;o t ,dwi t l ~tnd
5fort,oft. qt f OmiLted I'l'tnt~ late tota l .
"['lxc a .vorage m(,lecaalar weight , ca]culatc(t, fr(,n~ )t11 r(:.~idu¢.~. h "U|~O average mt) le~dar weig[tt, ca lcu la ted f rom t, heso, r(ssidues wh ich ,are ])rescnL in Lhe t ) ro te in in
l¢~ss Uhart |0 rt~aiduc-.~ t l)etcrt~h~cd a~ eysteic ~eid ~n(l cor rec ted f~)r ,(~0% rtwt)very ~fter per f i , rmh, ac id -ox idat ion
(Sc l~ra l~m, ~toore and l i igwo<)d, 19,~t).
I ' L . . ' I L I I , ' I ( ' ,X ' | ' |{ )N AX I~ ( : ( )M i~OSIT I (~N N ' l ' t ' l ) l l , ;N ()1 z f l~ .C l lYS ' I ' .~ , I , I , IN 263
A ~J~iJ,o-termimd ,mine ctcht deler~niuatim~ The fJ,-crystallin was converted into the. I)N t' derivnt ire t,)" t.lm me-t.bod of l~raewke, l -
(JortrnL, I'|arris and I,evy (1955). Two-(limer~sio, ull cltroll,~m)g,'al~hy (aec(irdin g t,o the met:l~,:~d of Levy, 1 954 ), of the et, her,sohlbh~" fraction of Lhe hydroly:ds mixt;urc revealed 2,4-dinit,.'oalfiline aml 2,4-dinitro|Jhenol only. The latter was verified by re.vm'sibh: decolorization with formic acid.
Chromnt:ogn'aphy of ~lm nqueous layer only rev(!a}ed ~-i)N[)-lysine and so/m: I)ack- gl 'O l~l td co lo l t r .
"I~AIII, E | 1.
Calculat.io~. ~f the appu.rcn~ s/)ec~fic volume of J,-erqsmllb~. from it.s omi~,'~ ncid composi- lion.
A tllint) n.ci~] res idue/ t 00 g
Amin, ) ac id re~/due ~,I pro6ein (wt°/~,) • ~ ¢× wt,',.},
Aspttrt,i[" m:id 4-73 0-60 2-838 Aspnrag inc ~ 4- 15 0"62 2,673 Th re, onine. 9_.5~ 0-70 - } .764 Ser inc 4- 8N 0-6:1 2,9,{ S (';lUt,~.ttli(" UOi([ ° 7"117 0-116 4.SI14 ('I t at La an i tic 6"4 7 0"t17 4- 3313 Proli,u: 3-22 0-76 2-447 G}yeine ,'b .qtl 0.64 2-543 A lau iae 3-45 0-%l 2"553 \ 'M ine ¢-07 0-86 3-500 ,Methinni,u; 3"(;.'~ 0-75 2-723 I s(~| et ll:ivt t~ 3-95 0"(10 :~-" 555 "L,etocine 5.5,t 0"1|0 4"986 ' l Jyrosine !)-2tl 0'7 l ¢5.575 ' t )Kenyhdanl ,t,~ 5".20 0.77 5-544 l<ysine .5.47 0"8:2 4-485 H is t id ine 4,-7!1 0-67 3-20~} A rgitxip.o 9- 99 0 .70 (i* 091{ ' l ' , 'ypto i f lmn 3-20 O" 74 2.36S Tota l lxMf-eyst ine 3-2 [ (I-03 2-022
Tota l 100- 16 72-825
* Ca lcu la ted f rom the re la t ionsh ip : wt (~g lu(aap)
res idua l n~qlee~dI,~r wMght glu(a~iJ) wtO/o_Nt l z ' N wtt'~°ghl wl~°~)asP ~: residue-/ utoJe¢mhLr woghg -- N I l ,
rosi(lm~t ~noleoular ~woight, g ' tu2r ' r - '~ i~u~'moleeu lav weight, a~ re.Mdua} molecu lar ,,','eight gl u NIuI ~{~'~i,,~ I [ ,)
The absence of mono- ~md di-DNP-histidine, and DNP-~rgitdne could be demon- s~ra|;ed by one-dimensiou~l ehrom~xtograp] W of the aqueous layer in the presence of di-DNP-histidine aud DNP-arginine as internal and lateral controls artd by the appli- co.tion of specific colour tests (P~uly- an({ Sakagucki rcacl;ion). A short hydrolysis (4 hr) of tile DNP-protehl with 11.2 N hydroeMoric acid in order to detect t, he labile DNP-prol ine, gave a negative result.
2fl.! A . ]". VAN DAS]
Finally, cystine or ~r half residne of cyst.ine, could be excluded as terminal amino groul~ after hydrolysis arid chromatogr~sphy of the DNI ' derivative of t~hc performic acid-oxidize.d protein.
These (L~ta lead to the conclusion that there is no free terminal amino group in fl,-crystaltin.
Carbo:mjl-terv~inal a,tino acid determination. ~.iNvo-dimensio,ml chromatography of the mixgure, obtained aftra' 16 hr of hydra-
zinolysation, yieMed glutamic acid as t.he most irnpor~tmt spot, but traces of tltreo~fine, serinc, glyeine and alanine were also t'mmd. The carboxyl end group was detcrmiimd in a qumltit~,.tive manner by enzymatic digest, ion with carboxypept~id:~sc A.
Products of the reac~ior~ at three time-intervals (~, 8 aad 20 hr) we,'e idc,tCified and esti,rmtcd by t reatment with FDNB. Sm'prisingly, very low yicIds of free. amino acids were obt~fincd.
GhtUm'dc ac.id appeared fi~t, but even at. a weight ratio (carboxypeptfdasc to fl,-crystaltin) of 1 : 25 a.ml even after 20 hr of digestion, ,m higher va.lue than 0-07 mole per mole of protein was reached. Traces of other amino acids, perhaps l)ro(htced by atttodigestion of t, he carboxypep~idase, were also fomld (0.009-0-04 moles). O,t ~t~e basis of these results it may stdl! 1)e concluded Omt ghttamie acid is the earboxyl- terminal re~sidu c of the proteirt.
The low amotmts, found in the quantfitative experiments, may be explained by asmtmit~g a proline residue iu the immediate neighbourhood of the terminal group. I t is knower that protine near to the C germinal end has tm inhibitory action on carboxy- i)eptidase A.
I t is noteworthy t;h.tt l~iekli, Ghazanfar, Gibbons and Edsall (1964.) made very sit,ti- ler obserw~,tions with carbonic ~mhydrases 5:ore h'mnan ery~chrocytes.
4. Discussion
The experiments described above show that fl,-crystMlin can be obtained by it combination of gel- and ion-exchange CtLromatog-ra.phy.
Th~ elec~rophoretic and altracentrifltgM afialyse.s indicate a high deg-ree of homo- geneity of the product. : 'he results of the amino acid a.mLlysis Icd to a molecular weighl; of 28,402 in agreement with the resuIts of the Archibald measurements.
This dat, um classes fl,-crystMlin with t, he low molecular weiglrt proteins,-confirming an earlier observation of .Rabaey (1959) from nlbrafil~ration ~tudies.
Based on the common assumption t-hat otfly molecula, r size. determine~s behav~ottr ht gel tilt, ration, it can be sga~d that 7-crystallins, fl~-, fl~-,/3 a- and pre-ec-ct3,st.aflin belong to the same eatx~go W a.s fi,-erystallim
t-[owcve.r, since-/~- ~md ~h-crystMlin emerge in ~hc rear l)a.rc of peak ":t (ct'. Fig, l(a.)), it is very likely thee the molecubu- weights of both proteius are slight.ly lower 5lamt that of ]?,-cryst,:filin. In agreement with this asstunption, ]3jgrk (1964) recently reported values between 19,000 and 20,01)0 for the moIecular weight- ofy-orystall ins, isoh~ted fl'om calf loltses.
Compar;,tive gel filtration of extract;s from ~alf-, cow-, pig- and horse-lenses (van Dam, manuscript it~ preparation) she/red that, the qua:at, itaLive prol:ortion of the compo~euts in the group of the low molcctflar weight proteins differs for the respective alfimals, a fact also noted t)y Rabaey (1965) with fishes.
PUI{IF ICATION ANI} COMPOSITION STUI)IE,S ()1" fl~.CRYSTAI, I, 1N 96,i
The f i i i lurc to find ~J.lty N - te rmi lml amino acid can b,: expMined by miltttoM/]g t.hat l lie termina l flllli.llO acid is blocked by lul t ict~tyl ~.rOllp, The l ) lorki l ig b3" li/~lcag(~ to ~t r~trbohydrat.r; gl'()up Ctlll t',o l.~xc.hldi.K.t by the recovi~ry of 100°/o in the amino aeld tl l l t l lys is.
1I; is notewort.hy tb_,lt. Bloentelidal, Bonl., Bene.d(st.l,i .'lml Wisse (19(15) inltde+ a simill~r observat ion wi~h highly ])urified ,~.-or.vatatlili. On f~lie oil ier hand, BjSrk (19(J4) fomict glycim~ as the only N-t~rmina l amino-acid in tit(; y-crystatllt~.s, isolat,.~l flx)m cattMenses, aad we fomzd g lutamic acid as N-terminal group in the major part. by far (if the fl-crystMlists ( iml)ut)l ished experiment.,;).
This emphasizes once moro i;tm eompl.exi~y of the [en8 prote'ins. A nalyse.~ of ea rboxyl- terminM amino acids of h.'.ns protcil is h:tvc noib ol'(.elt bc,zn l)crforined.
(.ilul:<~mic acid as carboxyl ~m-minal ,<l.mino acid ]ms not, yet bemi met witt, in any Ieris protein.. RceenrAy we tbund seriim as carboxvl ter ir l inM group +.ll" ~-cryai~ll l in (van Dam and ten Cat.e, 1.9(i6) and former ly we foruM t.|ireonilli2, i l l Sell'If) f3-eryst, allin.s (Vail D/till a-rid ~en Care, 1958).
Uiltbrt.m~at, ely, tile absence or blocking of tim N-tx;rminat amino arM, and the findilig of submotar ;tmolml;s ot ~ o~u-bo~Dd-termlnal grv, ips> make it, irnposaible rx~ answer t.he question whcf~her fl -crystal l in coimist~ of ore, or l;wo po ly l~pt ide chains.
]?rclimi~mry starch gel elcct.rophor(rsis ill 8. ,~ ~trea aim at p l t 3-0 did liot reveal more than oim band, suggesting t, he e.xistcrmc of ouly one polypcpt, ide chain. Itow(,ver, t.he possibi l i ty of S-S-bridge(s) between. 1;he c.hahts, if present, eannot~ hi., ruled ottt..
As for the occnrrence of fl,-orysttdlia, we timnd conll)onent~ in ext=.ract~s of calw.:~,~, horses at td pig le~mes with the same. electrophoret.ic mobi l i ty and occurring in the same gel filbrat;iotJ, f ract ion as bovil~c fi,-crys~,aliin,
l: lowevcr, immunoclcci;rophoresis of /3,-cryst.allin witl~ ant.i-chicken h.~rus m.ra did no~; reveal any arc, which might indicate t, lmt; t.his proi;ein does not occur in chick lens.
Coitoeridng the disvribut, i on ia the nd~tlt bovine lens, f l ;erysta l l ln wa.s fouttd both i~t the cortex and m tim lmdeus of $tte lens, but. the relat ive proport ion of f l , -crystal l ia ~o7:c altd y~-orysl;allins was much higher in the cortex than itl the mleleus. I:lowever, the total a mourie of tam low uiolecular weight: fraction was abotit 3 t imes greater in the nucleus t, han iu t;he cortex. In contraction witAl this, i t might; be worth wbih, ment ion ing tAl~tg Papaconstant, i~mu (1965) recettlJy fon.nd a difference in the eteet.ro- phorcbi.o mobilit, y of what he called t~ho <'),~erystaltin.¢' of ~>orts;x and mmteus. I t is t ikely thai; this is due to the. h igher ~tmounl~ of~q,-oryst.allit~ in the cortex.
AC, K N 0 %VLI'] l)O E~ {E N:I'8
The auVhqr is hxdcbted to Miss T. Opchmr, Mrs. M. G. d. vim Dam rsnd 3Ir~. M. (~P,11zejrI0.' i l for excellent technical assistance; to .i)r. D. 8t;hmhlt, Net!mrbmd.s institute for ])airy Re,search, ]~dc, for carrying out, bite ultracentrifugM analy.~e.~; to l)r. C. Mo~tIfoort, t,aborutory for Physiological Chemistry, St~lt~. >. Uuiver~ity, Utrecht, h>r i×-rf,Jrming the amino -ickt analyses li'nd the hydrazhlolyMs; to Prtifessor Dr. l[ . PA,>,'rn(~n,Ial ,Ltlrt Dr. ,l. W. .IJoot.sma for critical rein:iris% and l.o ~li-~. J. (I. J.lcidL, Mr. A. ,It. ,,'mi ll<~rs~-u and Slr. Th. ]~uiskens for ,skilled a,ssiatalicc in l)repllriilg till; llltlltti~c.rillb.,i,
! ~ E FER ]~N CES
Akn, bori, S., Ohns~, K. t~hd Narita. 14.. ([952~. t~ull. Chem. N<~.. Jap. 25, 21.I. :[~jiSrk, I. (196|). l~,xTll l"..'y, >. Hen. I, 145. BjSrk, 1. (19rJ4). E~.~tl Eye tle,~. 3, 16.
+'++'+ A+ F. VA.~ +>AM
|',+a,.~.Im;r~, ~. +++~d |+,++~*.++++r. s,. ,:.;. ([+++~+ ~. /h '~'~ ,,r. ~ , I~+L l+[+'.+.m,-n+l+~.|, H. , J~+it. ~,V. ~ . J~o+w+:tr4,~.L FL I,. ++M ~,~,'~.~m+. J.. H. <li~.*t+. +~++~-p+'l i+~'~+ l¢,++. 4...'+ti+++, ~.'+PJ,*+. +G. 3. ++.+uJ F~+/m~,jJ+ .J. '|'. ( | t~3J+ l'r,d+~im.+, .l~t+ix++> :|¢+d..+ ++~+d 1++:.++¢+l+++ +~+ 1o;+.+ <~+Id .~)+j~d,+r [++.J.+++..
N~+ Y;++k, P,+ml;++Id P,+bti+hir~ + t"+.+m+~+++>', +1+~ i+ l,t+m++, P., Ter+..rkr+. C. +,+~d ,.f1.+l.++. +|. + tG++~l++ ~'.+.t/.+ri+..'~+*.+'+~ 20+ 204. ++|,+ira. l'. t t~+~), +I.'h,~+i~ t::,,,-++,+a.y ,,f t.~plmolm. |:°r~+,':t~++-l.('+,++:+t. l l . . Hmr+'~, ,J, I. ++M I++++",', A. 1++ (t~J~k+~). Jlt+,~,+l ++ ++..( Ithjcj++ni,++d +.| m,+l#x,.+. V,+L :~.
+'d. By t}. Gii0k+ N,.-w Vor~ |l'+tPl~<.i~-t+ry++,, }+. "+.~t), Pr+jlp>~, ,~. ~,J'~<i }++~t)+~+y, ,~+, +|t)+'L~+), .,+tat+ J . ())'.~ttlvr++pn,'.+l. ~+ .['+.., | | , "~-+'~. ]:r+++t+~'*'~,l~+ ,.)'. ~.~+i }~+Jh~+.?, ., ,~.~. !l ~++0). ,'Jr+.',/+. (.Ji+lJJ)~++r~.+>+. lCi*++~+++~.+ )` 6+. +)++}J. Frmaq, d++, +jl., I++ha++'. 31. ~,+t+'t Wh~me. J¢. (it+~l+.. l~mlt. ,%',¢... i%.~+~'. OpAl]u+l:+~d. 67. ~+;, Fr~.r~r,'+~i:,,, ,J,, RM~.m,+,y, M.. XV/;'+n:~, lt. ar+d Kami,.~ki+ +~l. (It~'+~l). +.l+iG. ,1. lff~l~+~l~+d. 42+ 577.
• fh~fm"+it, T.. W. m+d M¢+r~tt;, [*+. A. <. ItJ,l+;h lli~+h+.+++ ,.I..RL ++~).+~. i(+~+e.l+-/X+. 1. < I+~IO_L [+~++,+L ,<r(]+. P~,~I+~.., 82, .151. t~---,'~,. A. t.. ~lt l&t) . ;',,',+;mr," 174, i~t++. .~,|t+xz,+Jt,, W., An++r|rarh, "l °. |'. +~+~+j J|++tb<*r+, +~+ J'+ +l~+j~l), l'+.d,'ri+ti+++~, l 'r,~. 19, 3"++.i. +~+a~mk.i. W,. |l~.l|.~+r~t..~. 1'. +rj+l ,;~,~++++m~h+ T. P. ~}+36A++ ;,It(Z++ [l+++'J~+..+++. llh,l+.+.+.. 92, +12. ,~te~l+.,+~++~+ 3?. I... a++,+.+ 3h,+h+IL K, ¢1+i+,~? 3. ~+ri++++++ I t6 , t4:L .M+++r'~+t +.-" , <+.. T.. t+|+U4+. ~. l+~.y+~++d+ (.+/~e+e+. I~, i+l. P,~l+++<~+++.+++++.h++,~:~, ,~. <+ 111+-;,~+, ]+~,;~J+im,. l+h+lJ~+d+...,i+.-~+. I.(~+ ,~J. +++~+,,++++~ml+~+_il~.o+.+, .[.. ~:t+mt+Jk, ]:t+. +:L a++<) ;'~+I,.,. lq. (lg+++~L .l+i.+.+++,i~n. lli-.l++++, o.h'.~.~ GO, ~0,', |t~+:l. ]~, ;~. Jr+It] ~|OI/'+P+, ].. ~1+~'[~)+. ;++~+t+'fl.. ]~+]tllp+. ], ]~+~. )~.ml+~,+y, M. {.l ~J.+4U. "l'h+.+'6~ t '~J'.',,m+Ly of + ;1+*'+~1. FtM.,~+'y, M. +li+*.~+t i:'r~r+,/+x ,~ !1++ l+i~+¢++7ie,+l /"f~+id.+. Uo.lloq:zh.~zn []r+)+&++t, An;.~tot+latn. l+:l:,,,+.'i+:.: + +
l~+~h}m.}ti,+,~ C~.,ml++ny. H.ii,kti, +~. l+., ~ ;++~.z+.+f+.,r. ~..:X. N,, +.;d+t~++++. ]L |l: ~x~.++} I++Imti+ d. "I++ { }+,.~t+.i.++ J . l++'.,:d, i."l+e+~. ~+9, l+.'m.",.~= .~+h~+x+t}t.+ [L ++". t~t.+~d~-++i (+~t++, (.;. (|~+J}}. (++z.~, Ji+++:e+Pd+++J .~?,, ~~'J2+ .4++h.'mid+.. D. +;. +++d t++r: t",++<.. ~+, t;+t'a+~), ("h,-ra. i+:ee..~'~'d+~+l 58.: 2~3,
;'+m~zh+,~+ [+, ~ t.+)+++;]L 1;6+~+++~..I. 7! ,. +~.+d,. ~+++-.~or, A, (++~++..~'~:+'++.+++im. lh~.;l~+~+, .d~:&~+ .~8, lg l . ++Fi+m+ +.tr l't. n+++| t.~h+~u~h++r,++ D.. ~'+ +lit+++++}+ +++++++l. +:'h,:+,+. Zl+ (~4!.1, T+++~t.¢~+a+r~, I{. t+ l ~+;~!'+~,. d,. l%y+. ('hi+m+ I~+ t ~._+l I. • m.++ ii~m+,..+X, tL .~t~d tot+ i.:+t+,.., f+. { |++~it, (',~+':-+. Wee~¢d++d e~. ++9~. v.+..++ l>++.m, A. Y.. ~+~+I ten (~'+t+,, t.;,.-i|+~-W+i+. IPi~+~+i,+. l++++!,j+.++..+I+.~,~ 121, !~{.. ~A+P~+lattfbr, 1.+. I." ~t~[.t,~. | l t , ;{<j+,t+t~,~..+r ++++ l~+';dr¢~ +'"l'+vq'+++++'y, ~,+'~t+. ~ V[ ]. +++l. |+V .~[. ]L ]+'+.++~+lt ++'1<|-J. '[~'.
Fki,,'dl. X++w y~++~, .Xr,~+.<-~++i+; PP+++ ~+~'.-. r,. '~Kt. '~+:++~+,,+ I. ~, . ,!+ { I tL+~O)+ +i%e.~+ t ++fi++~:~+~?," t:+f Gho,at+
Purification and Composit ion Studies of l~,-Crystallin~
A. F. van .l)at,1
The Laboratory for Medical A~mtomy ,u+~+d Embryoh~ly, Stale Uttft 'crsity o f l :trt'('ltt, 7'he Nelherla.nd.s
( IIc(..,eived 11 May 1966)
~),:e of the tow mtdet:ular wsdghl prut(,i)m of bovine, lens t,xtraot+ (lt,slgnal4.~ t~s ]J,..ery,tallhi, was purifie¢t by gel-liltT'tl%itm on t-;epttr~(h:x (:-76 alut chnmmtography oft. ])EA E,,%.'ephndcx.
The isolated proi<,in al)l)~mn+d to li(, |lolilogoneotts as jtidgtut by gtq.vlt.t:tro|>)tortmi,*~ ailtJ t,ltrae+~ut.rifugtzi an[Hyscs.
The nlolnr exq,inetion eoefti<~ient, at 278 mtt is 5.2H x IOL q'ha." ~ ' . *e . r r l l i na | t tn lb~o aefd d id ilOL react w i th 2.4-dh~itrotltiotx~bi.tz~+te. TIL¢-rt; is ovi(lt:tt('(~ t.ltat tit(: earb+Jxyl-te.rmbtal group is gluLtlrnie acid. Atnino acid t~,liid3,sis +vau perfbrmed with a. reoovory o f I00,II+.% ,)f t ht.~ dry we ight t ip l-hi+
[)Tt 9 t (,+jltl, '.I'tte rnolet:utar wt4ghL, etdeuJatetJ from the ftDlitio tlt+id corn+misitio+t im 28,402 artd i,
voltsisleid~ wilh the +rll[I.(['. tlc.-~':rmine4 l>y t, he approae|l-txi-oqtfiHbrium me.tluid. 'l'}z+~ proleili ¢ont,o.Ji~ 24(1 I+n)irit) acid +~.,sidu(m with the fol iowit~ (wmU~.mitiofl: Aapt l
Thr: St'i+ul (i|ilil i Pro~ (.lly~o Alait Vit l l i ~ill<-t.~ llcli io ][x, l i i i Ty r t , Plii.%t l ,y~i i ltisio h rg l i Try s (C.y,s- Stt): (Cys-- ,%'4 - (-"YSlz ( - -CONt l i )~-
]. ;nlroductlon
It, his ct'lssical work Mfrn~r (1894) dcn~olmLrated, on a pr<qmr~¢ive basic, thai.. (.lie. lens was composed, of three soluble proteins, ~.- fl- and ~,-cr)~ta||itrs.
l,+~ter on, fro'c-and ptq)t~r-elet;tr6l)hor(mis of the soluble proteins of Lhe vv, rtebrat:o let:s supported l;his classification (lies,~clvik, 1939; Frattgois, Rahaey artd Wien)e, | 95,t). Chromatography tm ion-exohaiigers ( Papaconstantinou, Remfik and Saito, 1962 Schmidl, and t(m Care, 1961, 1962; Specter, 1960), inmumoe]ecLrot)horesis (Manski, Aucrl)ach and f {all)el"c, 1960; Manski, I lalbert anti Auerbaeh, 19t31 ; ltran~ois, [Oabaey, Wiemu and Kaminsky, 1956) arid especially ZOla~ elecLrophoresis in agar gels (Frrm~ois and.Rabaey, 19.57, t959; Rabaey, 1959) revealed the het~rogeneiLy of each of those. fractions. By meaJm of micro-agar eleetrophorcsis Rabaey (1959) fourtd 11 fractiotm itt extracts of cat~le lenses.
In particular, i~he medium fractions were highly qomplex and 'showe~l a tmaeatred band in t.he pherograms:, in which complexly r~olved fractions could not bt~ demon- strated. However, tim slowest part of caruso medial fr~ctions, called Mt by" Rabaey (1959), tbrm~ a sharp band. Since the pro'~eiim with ittCermediat~ mobilitiea in ft~¢ and' zone-electrophore.sis have i tsually be;e~ hldicat~l as the fl-crystallirrs, we prot~ ~o call this e_~mponent fl,-crystalljn.
In this commurtication the isolation and some propel~ies of this protein will be report~q.
2. Material and Methods q'lie, extract~s were prepared f rom bov ine lenses as fol lows: i tmnud ia~ly af*~r s laughter
the eyes were removed and t rans fer red to the laboratory unt~r cool ing, Withit~ 2 -3 hr a f te r
This work was supported in part by grattlm fro'in the I)uteh Organization for Pure $~imatitlc ICtmm, reh (Z.W.O.).
255
~edl ..%. 1". VAN 1~,~,~I.
t.hu dv;.tt.h +.ff the tt~jim+d~ |he. ]+:n.-~,~ w~..re t+xlq~ed, w+LMt~:d wit.h cold phy~iu . l ,~ ieM mdi~v,, d eeapmdM.,~(l, su~peuded in ,;o!d w,h : r (,3 ml /g le..u,z) ~ml ~t i r r , ,d a t . .3 :~) rev /udn for !*~ l i t ,
Tho e, ort.ic+tl i~×t.rttt.t, t)bt~tinetl ill thi.~ W£ey ~s'a~..t e~.wtt.rifuged ~tt 7;3,~XJO .~ g ill a t lec lcn l , l~- ,qph~t-o l,, I ecdLril 'ugta fi.)r 3t.I mi l l t.o re tnt ivc t.ht: inst;htble, tdlJtttil¢~id, 'i'ht~ op:tlt,.~c~onl/ ,upern l l t l tn t wiu~ lyoph i l i ted attd ~Lor~d ttt. . . . . . 2 ( 1~ {~3 "
No ~iilTer~,nc.e, itl (:Iivotll~tl.ogi'itll~Ly. ~lgttl'+- atl+l ii~lttittlxo6|e~:f, ro ldtore~i ' t wet , ~. ~,b:~erv+,tl b+++l.wcotl I,hi~ lyop| f i l i z t .d a~i+l froMi m~tt~:ri~d.
Tlt,~ txt.~v l l t , ,V f i~rm of Sel?htuh,.× (I-7,5 (i-+harrta~t(Jill-Ate. UpI~/~i~h.t, Sweden) wa , u~e.d, wh ich g ivea +~ bt':(i.or r~ ~otut,/utx ~lxtl n h i~h,w elut io+l ~'~tt:t~ tlttt~ l+|to, fo rm we uaed pt-t:vio~.~ty. q'he gv~lilu+ ~yerv ~w,dh, lx ih O'0t .~[ 'rr i~ bufr¢.,r, p l I ?gJ l',~ "~ hr. '[ 'he fil~t~t gel p~rticle.:+ w+~r't~. x'e.m<,vlrd by ~l~~t'.~', t'~t~tioll atxd t.|d.~ procedure was r(+.pe~tLt;tr| fi-li t.imt'++. A rt+,r t,h+~ tinafl ttetr~t~L~- l.io~ the t.hit;k P+lush wa.a .,i~apenttc,,d itx ~hc eh.lthl.K buffe.r ~.~l+d dettert~t+t.+,,.i fur IP; nxiu. 'Vh,+.
l,~ttt'it~g t.~>~+~k 'pht0t~ tit, roont teltqu+vttture, ' l 'ho gel bed w+.as w+~,+hed ovex'~6~ht, wii ,h l~ufl',.<r ie+ the c - ]d r+~ottt (.I<'C) t.,, let, {t. ++t~d,ilizt,. The col+.lltitts ilse<l h++d th+_', dim,.~n~io,,~ t,t" ~.5 +:m +.+ l(~.) <~tix ~tit(I ,-,r ,I-,~:; c.tli :< i(~0 ~,~.ilt,. The . +ttnotmi.n of .+ample +tppJied were 1-2 ,t~tl 8-, <) g, re,~q,~+'ti v,: ly.
Ats elutillt~, butl't;l~s were t~s~| lt' l M '| 'r is I|(.'.1 i+tt p J l 7"3 |'or Lh,, tirol, ,..~"+t tilt;rat,i,~lt "~lul +~t
l,ri'l++lgbl. (xl p l l 7"8 (c,r p l t b.t3) wi/.h nmn'i++tti0.. Cllrt+,m~tol4r+tpbie :.,h+eld~ ~vl+re. i+t~+ttitt+t'<,,| by ttlt ul(.t'ttvi<de| del.,.'rt'tov tlllil ((l~,'i~-:t'~t't.] LI( I ' : . IL+ppmala, Swedetl)t:,+nt+e<:te+l w i th t.t vllopll+-I lu, r r t .etwder titl+l It'd hit.t~ u t'raet.i<m etdh.+etor ( I ,K IL ,'gwt~t|+,l;. t.~r t{+'+,+li.+ ,qwit.m,.rhtltd). l f ,) lh frt~(:l.iort ¢-oll+:('tt)r,s (q)++r~t.<,d (.,n +t t im, b,t:+i~.
[) I t ,rl~t+etrophotx~i~+e.t.l;r. <}'ht, J'rnt..Li<>lls were. c<+lI+,t.+t,e.d ill t,lto. +tl~prol)ri+;tt.+. lllltltllf+.i ', ~.tlpir vt)Iti++il,:+.+ det-e.r(tsir~i'<l+ +it+d l.h<..v xve.t'<s +Ii+I13"..+<~¢I ,'iti+l ]y++ph.ilizt+d. Aili¢,~+-<;xl:htt;tb(e (+itrotl)+tt<+- i.(l'ilpll, V ~,~,'l~ [>.+~+'ri~+~l +.,ut <;n .[)f'3Al~-Sel+lmdu, x .,%...~+O m<.+diui~i ~r;id('. (i,+t. +tt+,lbl,r +:i3[L ei~ p:tt'it.y 3- 5 .:L-1)- .'3 mt+.<lUiV./fl, l'tivt.icle, sizo I<~J--?,7t+ m0td+., t"Jtttrllittcitt +/II, t Jpt,:mhJ, S~v ,+l,m). Tilt. + i-lillt.~i'iFII W+I'+ Swotleit ft,r 2 d+tys ill t'.lt+" st~lrCilil4 hUffl'x' and tt io fille..+it. parl>ielt;s re l l i t lw, ti iLv itef.'li+itnbi<,i]. AJPl:cr swtdli.,tg, t..ho I)l,.]+,tl~-B~++tihad~+x wlt;~ ,,vJl++ht, ll t:tJll,+-te.l;uli<.'elv w i th
• 8.,+ wiiAi l i-~ ,sl N~t l . l lP i J i l'+. I N t lC I , wat,++r, i),. l +,+ N i i l t l l _ +iii it ~,+'ill,¢rr. ll.. w~iJ+ hPl+li l l | l t~ t.o p i [ "" '~ +ill{f wfiv.lied w{i.+h the st.lll'tiillg bitff~r.
' | ' l i t , eOJtltilliS {tiilFion~il)llV.: 7"~;-..2lOt.+lll)"livel'¢, ir)<iliirt+.tt..litid sefli.tiieiit, t:+l l iy grl<.vlty ,,it. t'fltTtiSi i=t+llil'ie..llit+lll'f++. <]['tilt le . l bed w+t.~ st. l ibi l ized hs" W£t+t].ll l l~ .i1+. ovt.+rliighl; itt.-I'~C wit.h flit, P~t.nrt,itig bllf]'or+ Thc'. ml.ilqlle.'L V+i l ' y i l ig I-i£q.Wf~.P+ti 700 i t lXd +e.7(7)II1~ i l l i~+'.l)>trltL~ +.~.xtt~riirxt:-rti,~+ w !re di . ' l .~olved ill l t l ld lti~llv.'+t:<! tt l i t i l tst, t;tt~2 ~t l t r t l t t 7 httlTtr.i +"
The olilt.l~Jil ts'lt~ eff~,eli,d b'r it di/~COlltitll+loii~t ~rti<il~;tit. (i it i<tni+: .~Lr(:iij_~.l.h ul/<l p.! l ) "rh,.: t ' tdlowilt l t ['ihoi~trliilt.e tlttlGi++:r++< were eltil>JOS+<l ( l ) O-lIO,~)M id l i~-~; (~) 6"f)l ,~t 1,tl. 7. I:
(3) (l+(.tl. M p l l 6-8; (.l) t)-f.l~ xt p f f i3-8; (5) O.(l~l .~! p i [ (l+~4 t-(i..l .~I Ntif~J. :+% (i-I lit.~,i I , ] t )TA ,~oiut ion. brc>ught tcl pl i 7-3 w ld i Pl l l it l l l l i l ia Wits lid~iu'd t<~ I;ll tliilrtt-'+i>~ --rtL,.+ frat..t.ic+ll.~ tilt<.lei" l,hO differecil. IW, ak+ wt-re pooh, it , ~lii+ly+ed uil+t t.vophiti~,e<l. Asz:,r-
t~it-cii'tq>iiort,~ds ~,,,'~it~ ipi~rfort¢l~:d ll(:v.<~i'.iilig t+.+ l..|ie Iiri>oodtli'i, of lili'it.li;1 " (I,t.).rtt. ~'') oil ,<,!7,t+'-~ ~i" }7 < ' I l l - : - f t i l l . Th<~ r,.,t~li.i~',, lilobititi+'.++ wt:re e.alcu}at+>d wi th t.t~+.,+ li~iel-hot.t Of ~IVit?ll)l~ (l~,IrSt)).
\"+.'.rl.i<.~+il si.::ln?+h gel +,let~IrtJtlhor,'~sh~ w+l~ earri+ed ~,+~t +l:.~ d,:,.~+erib~,tt by Stilittii<~>+ (l~,t.">+,i). "['|it+ 71+1 I~ufl't.w xt'~l_~ O-(i(.+ .~1 ' l 'rbg t.iLi'ltted wlt.t+l h~icio. +reid t.<l pl-I 7.t; i i i i l | (i,,~t M t>oril, :lCid: t.itt'.<tl+-,t +<, p t l 7.+I w i th Na(. J l | , wll~ +.,mld,Lved (,t:" t,ridt4,+ l ltiffer. /4]ie+:f r<lllli,~r,%~i.+~ i.,~,+k" pl+j<'+.~ +it. +1""{7 tl,,411:i|l.'f f~r ii+~ • t~ hr .
3 ¢., ii~ a I.r|t'+'+'~t'.+'t+l ri|'tU.~+LI +~t~l++l~,'m'S ~,v+.,re. Illtld," i+t " +: " t ' fLyw6 ;lir-<|r{t'+,ti ti lt t'+tt'Plill'iftil{<, 'l'tl,: [tr< t+~i+~ wll~ ~ti~:~.,tv,~l hi h,,~ri.liLttr;~te tllift'+,.r ++J+ i~|I 7-11 ~ntl ~dju.~l.~,~i w i th N:.tl' l li> al l i,.mit- .~tlei:ll~l.tt t , f 0,T i i / t .
[ b,t ~, i .no] i ,v ! . l l i l l + %,+'t.il~!tt, vi'::i+~ ,b+t, , r t lx i r tn , t tti,, ftT~+;~ i+llqtr<'>a<'ti+t.l~~~.<'~|Tii l l ' l lt+tli<~il-i 'qil i! i l .tri~tP+ . l lt~>lti 1+i, dt° l , . i ' . l ' t l l{ l l l t i{+it l : 4 l i t +~it'lrt'l'f'llt ro|.+)r P+p+~_i:,f].~ xvt~rf >. iitl"ol'l14~+,~+l,~l i l l t<l ~t Ti'~+tlt lq.<ii i t:{,,t {'[+t-~ttli.tll i ifl+ i ti~'.~t+i)+
I ' L . ' I t l I , ' I I * .A ' ! ' I ( JN ANI ) +~t)MI ) t J s l ' l ' t l )N ~ ' l ' l ' l ) l ) , ; , '+ t+l ' IL ( ' t IY .~ ' t 'A I , t - t , '~ ,;;~',7
Ami lu , +u:.id-itti+d)',~+'+ wi,r(. > t,(.~t"f+,r'iti:->+l w i th )t 'l',,t.hilh:<+s.i i l . t l l l ix( ' t~l"llt ;-;l~.t*.t,) I.+"l i it-i +et'lttlt+, ~ I.~) t.)lt' iillHi.lll')tl ' I l l t'i~,;,. , l l id ,Mt,l't.~ (IW(iII). T, , il~+i,'h] l ' l t , J~;itt~. ~ )#i r i l l , ' I.iil'+t".>t.r'¢ |ti~J~'. ~" :~ 1 i'tJl'>ii~liftt.+~ liuff+.,r p I I E,..~) was ll~'.>tl iil.'(I.t'iti| Of i ~.1 #,<iltli l l l i lt+'l'i';il<; ),iit,l'i,r i , I t r,.D (M,~.,i#~J,-i~_ [t¢.~!'.~4<)1<t~1] i~ l i l l l l l i l . i t l i l ' l l l , i i ) l i ) , tl 'ttr,+l. ' hydrv ly~i~ tixl~,.,.,~ w,..rv t~.~¢d, vl~.. '7.t, 1<~ +,)i~l ?:~. l,P
Tif+.V.llt.olJttitfi lt"lt#i t l l>l.e. l ' l / l i i i i ' l t ],..V I. lte l)l,~l.h(l<! (if Spi ,>~ +)l)il {'litl i i ltt. l '~'; tl'~+.l~q ,~,1 h~: ttl~, s l i fq ; t i ' i i l iho l i l ' l i l i~ l .F iC t i l i , l .h , id t;'f |J+>()tl'*l¢ili si l i*] /~tlf..,rtoli ( i l L | l ) ) .
'J'}lt~ t~'iti.l~r-(-oixl.i)l l l.~ <if i.[l(f, ti~,<,i.elll r i , ; lut it . l f t~ wt~r+2 +b-,t.~.llt~li),,,.f l )v t t r )o i l t~ I.*..* v-lili~l+-l~t~t weight, ~t l(.)f)"(!.
[! lt i '~tvitdt~t; ~+lit;(:tr:~l )~tiiil}'~,ii+ wi,:+ It llil+." iiX" l it l ' . ' i l l . ~, t,f it tl;~w~'rtl~il~ I ) l : ,~t,.,.'-l+',qt)t,:t(,. I l l i ' i .( ' / ' .
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~- l . ( ) f t l l l J l i t l ~t/ . ) l l t ) ditl4~.ltl l i l it i|. i+~ill~ V¢('l'+' l l i l l i l i : ~.%'ilt~. ",~-,:!l ltlliilf~.,t!fit~,i~.,1,,,lil.+,t*.+~ Jit.ct~tll i l lM. i t , lh+; l i i+~i| l l l i J <~f l ,e* , 'v l~,.i .(b,m'rib(:+l 1Lv | i ' t ' l i+! i i~+' l - ( ' l ) l i l l i t , l l l l . t ' t l~ li!l<t 1~ .~, %: i lit,#'.':lrt
(.~leil¢,~lt ' t ' l l l t t(r(+ll l i C]~'(~'/'lUIIUli.i+'¢i:'I tv,,r(, tt in. lb- wdh <'J l i l ) , ,~cy}~,. l l t i~|:t~, ) t - l i F l ~ (b,I ~ttl i l i l l t~r t~.tt~.-tl~lf+() SiI~i+lt+ (] Jt i>i ' i l lv lt l ( ' , i i l l t , , i t i ) ' , S t . J,,,~il~, 5 i , , ) : l - , | , , . ,+'ttl>,.,t hv |l+lt-ri.+ i i ; r ) l i ' l i k , ,1 .{J i ) i l l ' l t l t:L .tll, It t.i.r),r)) tl.I)ll |).y .1.tl)' ti.vd rli2.tll+tl.?':4i.~ tit+ -l, ti, '<i <'(" -:% k i i l+ , , t i . { l l i i~l l tt-{i~l ~'fll'i~li'L ( I ~.).'l~ ") li.q" +ll,m'r' iht0d i.)J' <|++ I,;i l,t+l.xii, T , , r l f i l l i l l ) t{ , | l i t t - -~. { 19+.=-li.
[tj-vu s l i l l t t ty l i l :yt /2r't>uf+-~ wet(, ttcl+'tl l l 't l i+,tl I,V t i l r : l l i+)* , t ~'sl l l p-h),. Is, .x,, lt , :-t ,~li+l-+~,,),~., ° tlcc+)rttint~, lti l ' Ill+'1 I i , - t ~,f i+,.l.,. W l l i l , . i~ll!i All|"llt~t-li (1 li.')tt.I
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(v~u~ t),Lm a.n~l.t~,l (!~lt.~;, ]~.)tiG)rt..w..)d,.,d t t,;Lt ~,.,.',rV.~,dl~L ~:c~lrr , .d ~l~ t I , ~ ta~l t~r~d,,t~
[*' i~ttr( ' . | (}.t) ~how~ iI / ) ' [~ i~, J ( ]~.~Lr i |~i t i on / . : i l rve ( I f 1+2 b[ ¢)f l~<:~'i~! J~~l.~ ~:4.~l't~.~ ~Xtr~! : f
wit h ")ll }ntemml dia.m(:t(~i" ~H" ,-t.P} t.!t"i+ ~'~'t~e h)ttd,~d w}. th8 g of t llt: ~.l~ti,g'.. +.~xtr~,"t.
w<~.rv i l fcuti l ld-t , : i .y r,,~ol~n:d. "J"jtts ~,'.h p~:.~k m~dl l lv ,ti.m,i>im;,r~:d wh,'~, t.h,; l~'~,~ ,,xl.r)~+.'t
ub+ct.r,) i ,horesis of th i s fr+~.t...ri~)i,, i~ fl~,:l, lil.s(~ t~otcd l~V i~ i i rk {i5,3.1.1 .~fter g,-i t)|l.tatl.h.,~
))}g,b),r than i,i. EF;O nV.~).
r~tri))" du)u)tt~d/~z, J]~ at~d ~,+er'5"sb~dliri, aud pt~:.a.,.(:ry.~A.,tllitt ( l:qat~.: ! ). I~x~ttliin:t(i()t~ (')f tit(: Jront. iltl'.) back purl. ,.:r |<,ak 4 ~how,.,~t tt~ltt o)~e ),.-c%°stia||il~
I.y+.)~ i l ie ~l-(;ry~_t<d|{t~, ttt~t[ i)t('.-~t-cry~t./~}.l[n +.-.m(.'.rgr~ iti ).ho fr,.m(, par(. at ;d tt~at ~.~t~r~<J ?,i-ery~t.;dli)~ c. lncrge ~ll t:.h+ +. i.qr(:.k |)+~t. cif the. | )~tk ,
i i(} ~. t)f f,.t=ez(.~-dt'ied Icn-~ oxl..t~+.-.( )'Jold,~..! 8 ¢.~f fra(,,th)it .i "tit al l . '|'tt<~ )~.)|~.(| grott~,s 4 ~v,~r,, r,.!fih~++.r¢>tl oi l ~el,hadt.,.x t . L75 . .An +*~)),)wn in |'-+'if+. i (b ) li~,~+, i+~)i).++ tma~; (fr+e.tio)i ~) cmerlf<:d ill. the mtm+s t~3,~iti.ot: +m it* tit,:, l i c i t 14,,t filtr,+!:Itn,..
~t.are.]t gel eh~ci.l~)i)l)or,s~is o f fr~tc~tio~) 7 r+~,~'c-aled ~i. I x~ i i t | it: l~!',~c).~+;ity {lq~Lts ~.t, A.~ ?tll.tt/,y O~i l.t lili/+lJ.! Ir.~'JttJ+'t [~e tfemc, n.+tritt,~.,.|; )~i 7-ctv.+t++)lht (h++id. i.-P,)< H,-'cC+'+t+xtti+:~
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nol+,~lt.i(m v~-ii, l l rov i . - . i+ ' , i~Mly <h;. , . t i~tt, - I f+ir ll.f~:i't.,,llc,,: l tUr l .+~,~, 17Jr ht(:J~ (,I' ~tn'i" ll,.~;l',.lt r io t i i (~ , l l l : l l i t l l t '~ , ,, l l | " i.h~, ~ l~',il~ l~i'(~i l: i l i .q.
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." : . . , ~ . .~ . :~ .::~.~',:. ," .~.~5";_.?+"~:~ :~ " ~ ';.' ,:i.-~~++:J~. ;r.;~' ": . : .~ ~.+'::.:++~'.::..'i~,"~. ;:~ .: . " . .+ . ...:"77.1 ~7:'"<". '+} :~ .<:','+~. ~: •
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~ i~i~i ~ !~!i ~ ~i~i!!~!'~!i!~!~!!~ii t'~il ~'~'~i~!i~i~!~!i~?~ !~i~!i~!i~i~!~ ~?~:~ ~' i !~i~i~i i~i~,~!i!~i~! ~,~,ii +((ii!~ ;I i !,ii~. ~, i~:~i: ~!i +,~i~!i,+~i~
l+J,+X+l'~ "2. ~J~1+';'lJ g+'l e]v '+.r+,l>h+,f'~.+.i+ p;,tt~.rt; +,I h'+x,t+t.;+, 2 lc,,l~+ l.l+t+ l+~rt+|tr+'Jl ++~i +'~.I~<,.++++:+++~ I[+'+ +w +J ~',).. ().t)+'i..+,+ ~l'ri+ l+,~l'++[.t+ ~fIti~'i~t' ]J|| 7"t~; ~ \".i'+.tt+i; IT',++ hv ~.t-.I i I+ i.,+tj.+h~ +.+.+~it:ehl;+~I i:+~+,' .. .~ v..,.+. +.Kq h+h.+~ .+t'iI+l ,tPi:++,lr++. • hl :~ek.
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t~._., ~ -:. O.(~l ), r,::.~-p,:,.'.l ~vv]v. |h ,n3 in~ ~t m,,t't, d,,l initiv~. ,'hara~'t,'rit.~Jio~t. they ai.t~
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Th," th i rd t,~'~K v.. , t~dn~ ~Hm~,.~t Imt'~- ' ' lm, tdlit t" of 0"31..5 tl-0t ~:.~,ul}i b- t',i~:uI**t,-,t. Th, . f,',tt,,wit~t.1 I,*~,k, .t arid 5 ~.t~*t ,hw pr,.~-
,:,,~,iRm~..m /t¢~:D-~l.~dlin. wets l'otlll,{ w~t.}~ ~i r , . lat iw, mid, J ibs ' ¢~f 0.57-.~. t~-OI.
( re l ' t t ive m,'~bilit ,." ~ff 1 <1:2-i.0-O2). ., {-
~hc ae,lu¢-'m'e of tho mohi l i t i<~. ~.~ c ,mht },c,~.xl~.xl~.-d. ,.x.i-el,~ wit h )'i- a**d ~,-cry::tidiit,. The n :~s :m-why, i~ n~t t:h.~ir.
~*ppc.~it~'d ~'ith the ot)~,.'-r-bttfl'er.~.
:t~;tl <%. }'. % 'AN I JA .M
/g /,', '# r . ! t r roTv ' r l ,'.v
t;'t~i" f lu - itli, r:irh>h~!.. / t i tsor l i t . i i t t i l i le; i , - i t lr l , l i tel it , - i s+',ll-t're<., l y t~ l , t l j ' l i ze t t pt'~t~,.il i was , t i~,,+,dv+:d i l l 4}-I)t),¢1 ,M ] J l i osph l l L f ; tmf f<! r ( t , l l ~4.2). T i l l , . l i l '¢ltt~ill (:t)lXl::i-lii.l 'nli, ili +|i'll.,.;. 0,9.tl:i.~> (d ry ~vtdTli l .] , ' i ' l ic; >~b~<Jrl)t,i,:m f t i l4 ' t : i . l ' i l l l i rev,~;th,d a i l i . f4x i i l i l : l l l ill. A~ l i l l tt , ! l i l ( l l i l i i l l l i - t i l l . t i l l ll.[, ~')(Ji lllt.z " ' l ' j l ( : fq>t.ic'uJ ~h,+lis]t+,y i " tLt i ( ; i i ] . t l . 7~f J i i , / . , i l j . I , . 21..~', ,(17, 1,rti,q. t'l.~llil~l l¢.l Iw~ "2" 17 ltii<.l I . lt[s %'lt[lit +, Sli~[.(tr:st.s Llirtl l ri.u<~l+,ie +ti:i, ts I¢l'l, ~tlt~(, i l l . . ' tV i l l i i.he. ;t l l l ~Jf <| ry x,,-t,ig, llt. ( l r>>l . ( ' l ' i i l i i l i t l i l i l l l ,+ i t i l I/'5 'z 5"{ V I I I I I< ' , l i t ~78 il/#X, t t f + [S ' i i I t l i t l ill.. 77(1 i i t tt i I l l " 1 .~'~'1%%'l'.1"t; t : . i i l t+ l l id i t * '< l_ I I ' [ l l '| 'h+" Il i i l l+ti" t ;xt i i l t ' . i it>i| t : l l ,~lth' i+'t l l , it++' i i i l . ~7~ i l l / t , e i l l+'t l l i t l . t ' l | ri.i>ili ih~, i i l o l t . ' c t i l l t r we i j~t i t ( , r 2~- . t l i7 . i,,1 ")-2;"1 .." |iS t .~i 1 t'zti I
' ;4
.- I-'..: '" ~ :77 I+-
1 ~ E : ,'. 1 i , L
s.! l. ; ! I I I t " , L [
I , t . J ~ :' : I I ~ + ~ - + ~ ~ , : . L ' ; _~., t ~ i . i = !..+,: f. 4",,.:.'~ l):'~".: I;.":'~[+ ,t.';.'... dOOr.- ~ p'i-;.:S" ?. +'." : 3;;'(~:) 3(b(~'O
l ; l ! | l t l i i+- ' l l v t . l l i+ . ln i , t ( r l ' : l l
l,' lfl. 3. t ' , ' rhr t , i t l : i l , , i . ' : r , i l ihy +~f t>:,~dt.d fraet.ir~l+-. 3 +,rx l i t ' : k IC.,'4:,I,had0:+. [',tl>+'riln+.,/It.a! <,+:ridili~>ilt% te l l .
As, ,+ui l i i i :g t.|illi. +~ae.ll L.vro,.-,yl i',:si<lll¢, g ivvs t, , :<.Jni.r i l ,ut.hui , , f 1-3.1 ~ I l l a i~, K ~.ltd t . ry l~- l .opht i l i o r F~.FIE)× IO :/, wt:. t'.lit(Flilitt~,d ft %-tllllf.; tit' ,1.t32 ;~. l i f t .~l i ('.1!t .I t 'rttl i l l}ttJ 1.li<,+4iil~" +i!!ttl t r .V l l l .o l l l i l i t t i :Ol i l .o l / I t i r l t t l ; }+rld~..-itl, t (i l iD.({ f'J t'~"ai~ltit'S, res l i t : ,St iv , : iv . The. r i l l i<, /<~,:*i'. +if I . ( i7 i~ i i t l i lt ' , t'llli+~(<t (tt'tl~t, --- i ' 1 '3} l 'Otl l i t{ If'i," %li'etl i l~lt' i .r l i t t le7) f~Jr e~+-:t.'~l':ll
i~f I" it l l"
i ' l l . i l l ' l " l t t ' l l t , l ' i l i S . Th is dl flm+mic.~.~ bt,i<ween i.h~: OXl~i_q ' i l t i t : t i l i l l i l l i i ] t t it" l .het ) i ' l~ l i l : f i l v i i ll~t {:~, n l,+, ,+X ldn i i led t>~" i l l / ~.+llhil.llti~.(.tl a t l s t , i l l{ i¢t l t {>fr the l l .rt) lt i l l [ ic, r#.,.4:+.tilt,.s tlS :1 r tmul f t~f l.[tl'.ii" t>l'l.'C'f+Ulll'++~- i l l t | l t~ l+r l .~[t , j i l .
[t.I¢~&te~,i,.lr o.'.~'i#]l~l dcl, 'rm i,~ll.7<m.s
ili.l.o ~l. "|'raul;l',tat~ plr~t., a.l'~ giv+~lf in [~'ii;~..t. As Be.e.ll {I~ tjl.i~ |ig{t:rt% t i,t~ '|~l;alltttxtt.rl p]ot. i~ l im~u, wh ich i~ charat:t,eri.~l ie. t'<,:" ~, h+mu~gmleot,~ ~tmc:i,,.~.
| " rmu lho sl~,}m of t im r~14r,'+'~'~icm 17m~, dmcrm[m.d t,~" rm..ans cq' tht: l,~,aa~, t~.qtt.<~l-,.,~ vlmt.hod, the z-t~s'+::ragc t , ,dm:od upimre+nt, lnt ,h,eu[ i t r wei.lcht, ;ll,,~,, t l.. ~p), t'uit by t'l~h.'u-. lat,w}.
+<ka+[gt~.it;g t~r,,," i.t.,i.t{iti+..ti,.~+, l imit, un<:l ~',+ith ~t-+uld+m:l de.vial i¢,ns e.+ilt.+ttl++tutl tvil.b gx+'+,'-2) d*~gr,.'+~.~.~ t>f l'ret.,|o**i..q, v<.dl,, :. o f (O-";I~:~-Ci-(K~} .* iD "l xv~rs l"+~ttitd ft,r t hi++ re+lm':a~tl ,m,iee.~i!i+tr we ighL
T im tmrt.i~t} +l,+:ciih: v<quilt+.: {+<') w+ts +:+ilc~tb,++,+t +,t+e,,nlin g t,.~ the nmth,:ul cA' ('<.*Ira told. Eda:d l ( 1'.)-13}' l't'+,ttt_ thc tt ram,.+ i~ch[ t,.++ml~osiLi<m, t+'or t hi, sluu-+i~ic x.~ltltlt+.: <if t:.ye:t ill,: t.hi.'. ¥ i ihm o f 51eSteok in +til<l ~l .+lmiit i l l ( I{ jhO) iv+i~, u.Sedo T im c. i~lcul l t l . io l i .v ield~'d ,l vt ih.u. l+l|" (}+7~7 l+ i t i t ~ {i+t;i~ T t l t j l i . ~ t [ ) , 1..)4311•it.%. + llt++l~,Sll+trtlil.t,.~ill.8 t~,t 3'a( >. U+i l i fg it. t (t l rnl Jl)'C:llot:~l+.+.Ler,
.i l ' ( ' l l l t t. hi,*t41'7' d l i l~ ;+ t : i lotOet l inr we. ig l i t +.~f 2SOt.r!i ..:+--l(tl~.i w+L,+ ~ttvi~. a '¢~lim'. of I'tRS2+, +" +;alc.ul~ited fro" ~J,-t, ) i t .a l l , i t .
l'I'l{II.'lt'.A'l'lqlN ANI= ¢ , : t Jh t l '~)H l ' l ' l ¢~N ~; ' l ' ( ; l ) I I . ;~ i~I: ~, ~'ht'V,~'l'Al.l. lX ~I,,l
A ~,~ ira> a('id ~:(',m I~'it i<,n 'l',tbh~: I. rm."x~r¢l.~ tim mnim);~<,i~l eOmlU)~iticm. ('~.mven:i~vmlty, tim v,tim, s f¢,r tt, r¢:o..
n in,:, se.ri n,* nntl ;m~id,:.-N | 1 ~ w¢,n., ex tl.t'uI)l)ll| 1, ~(! t (1 ~r~'.l'l} {i|'~ |.(~ t¥t" hyd r~.,lyui.~. F(~r v~lmr, ~tll~l i~,.detl~il~(! t.Jm ma.xbtml vtdue:~ of 7"2 hr h)'drolysi.~ tiara, w~,r~, t.~ken ~m,l f,~r t1,~,
I)upli(.ati:e r~m, lyse.s r,.'vt:uI an ~v¢~r~tg,. meu~ duvi~tio~, ,,f l'*.i.. 'l'h~, tryl,t,..,phuv~ ,:o~lfetl l , ([,~ve.rluitm,| n.~:t:~.~r,]il~,g t,) ,'.q,.l,i,~.~ ~ ~,ut ( :ham|mr~ (t9 i9}, ~tgr-,...u.w.,.ry wol] ~'i~ h
3.38 w~:iRtii. ~.'-i~, z'e-~l"<:i~ivel.g, t~,,:uus,' the t'(.~r~m n~'tb , - I i,~ v,-r,, re.li,d)h~. ~v~, ~',tteu-
Va~,I|I(',S, 111',' g.~'l',l { i l l ) " ,'X|.r~i})~]lt, tq:~[ (Ill{| l.-jl{ =- l l ta lXi l l l l i l i t X'IIIID.'.'4 t't.'tHtt l | | , , {.ltl"~*~" l~w|r<,lv,,b*
i
~ / , i ~: ~
~-'~ ' . . . . 0 -,~'~ ~, r~ O. 3 t . '0 :"a , . . . . ~a,.: .- . -
o.
t]me~. T im :era.~vm'v of I0~.).9'].i~ ,.>i~ u weiu~1, b~i,~ provid~,~ :~ ch~r:k (m qhe ~e.l i~|hy ~d' t h( ~. ve~n|t.~ :~n¢| in ~.ddi~i~m il ('.~u be co~e]j~,h,d theft, th,, pr~.*~-~~:,~ ~f ami~a, ~ '~[~
mob,cuber weigh( (rom the Arehib~dd me~¢~r~:n~,rc;~.,.
i~.d,-g~al l}l,~It~])~',~'* are given.
_n , . A . 1.'. VAN I),%,%I
'['h(: l>)'(,tein (:~mtains 2,1(5 residues. Since l,he, differc~u.e b(:twe(,)t (u.sp~r*.ic+glut- t)mic a(fid-.'tmitle) and l.y,.sine+argirf iac+hist. it l in(Q is 12 l:,t,,s'ic g)'OUlm, one might expe.ct, ,~t slight, ly alkalim., iaoionic poiut,. This expla ins t, hc slight.ly cathod ic movemet~t. of t.he prot.ei,~ ill the, starch gel e[cctrot)horesis a.L l)].[ 7.6.
¢ • I ~ l .h. t, it.raLion w[t;h /~-hyd.roxymcrcttr ibenzoatc rcveah;(t 5 S l l -g roups I)(,r )nolo iu (.he ~tad;iw: protein tat, pl:[ 7"0, A s l ight ly h igher w~tue (5.5) was obl;a.hacd whe~ (:he twot(dt~ was <.iet~att~ra~ed wi(,].t 5 ,,a gtt~,nidin(;+.[[(31 bt:fot'c ~reaLment witch p -hydroxy- mcr(:.urib(;nzo~t(~, a~l(l Lit(; opt ical (tetlsiLy was n~casured af ter 7[) hr. Thu.s it alq:)cars that, (;he I)rot(:in posse, sses 5 free su lphydry l gro~)l)s ~t~lti p robab ly '2 cyst.!he resi<l~(;s, I lowo.ver, this has ttof, as yet; beeu e..~tablishcd conclusive.ly.
TALH, t,: [.
A ~, in o acid compositim~, of ,B.~-cr.ystalli,g
Ami) )o ~to, i(I res idue
N(:al'eS(, hnLegr-~ I
Aufitt,) a(; id Agmm~t~([ ra). :ktnitl~) t~cid number ,d re.sit lues/ Mit~im)tl o f ros idue~ L :n lcu latcd resi(htc.s/ :~mi;~o ac id ]00 g O1' lll.O[Ct~'Llhtr {'(w 2,~'0110 g Ittolo(~ular 28.402 g o f too' 28.402 g pro te in a we ight ~' o f 1)rotcin wcighl , p r , ) te in (>f T)rot.e.i*x
Asp~tr t i c a~(:i<~, 8 -9 J 1292 '22 2S424 21 ,1)~ "22 Thrco~Ji n(.* 2 .b2 c ,10 t :2 7 2808.1 7 4 )8 7 Ser i n6 ~ 4.68 (" 186 1 ! ;3 27.(~ ] 5 ] 5.2ti 1 ;5 (}lt:'bnrnic ac id 13"S~J {)30 30 27900 30.;54 "~1 Pr olin(; 3.22 /tO 1 (l i) 2714.1 !J-42 ~) ( . ; lye!no :?,-96 144 1 211 2g,q2[} 1!)-71 20 i~. h~n i tw. 3-45 20fit'l ! -I 28S4 f~ | .'|'7!) t 4 Vtl l i ~t~ 4.07 d g-t 36 12 o(~,1_3,. ")" ") ! 1 436 1:2 514;tla i(,n i nu- 3 .ti3 3(5 t 4 .~ 28912 7-,'-¢(¢ B [s<)le,~ci)~(; 3.95 d 2~(;5 10 28(~50 !).!) | I 1) {.~.u(:it~e 5-54 '?051 ! 4 287 |,l 1:1.~:3 I -I ' ' ' _81 , ) _ 16 -1 ° !(~ 1 .')'r~.,slnt~ ~,t-2(i L71;2 111 ,, ' t -) - | )| Wily J tl.[tt |Xi t lt~ 7":~0 ".2)044 1 -I 281 i I (; 1 ;{-c.){) 14 l .y~i lv: ,)-17 23.t3 1 ~2 28 11 (; 12" 12 12 [ i i s t i~ tiu (, , i -711 °811: t 1 o '1811' ]0 9-( . )2 l ( i Arg in iav ~.)..~19 151;:[ 1S 2813-1 | S. 17 1 ,'4 A=ni,.h:: at~n)t,ni:t (1.39) er 115f (24) 27672.1 2.1.1i8 1251 "l'ot.al h ) t l f .ey , t i ne 3.2 ! i 3 178 .q 281)(.):2 S-94 9 'Fry pLopl tat t ~ 3. :20 f 58 t. 9 5 290.1171 4 '88 ,5
'1"< ,ta ]: 100"9-1 28402 ~ 24,6 28:167 h
5]~,~tt~ res idue wr ight . 116
r~.-;idue n~, lecub~r weight ) / . It) l) )) Cttlouh~Lod fr()m tLe rela(. iofta|f ip, of t lm!no ae idres idue it~ protcb l "
~" Value. exbrap<)httt:d, to zt:r(, | . imu f)f hydr(dysi .~. d Vi~Iuc o f the 72-hr hydxo lys t~e. t~ A va lue o f 3"38 {weight. l)ur t~()tat,) was ol)tMrtc, d by Oa(, Sl)ectropht)t , omotr ie ,nethod ~,f ( ;o t ,dwi t l ~tnd
5fort,oft. qt f OmiLted I'l'tnt~ late tota l .
"['lxc a .vorage m(,lecaalar weight , ca]culatc(t, fr(,n~ )t11 r(:.~idu¢.~. h "U|~O average mt) le~dar weig[tt, ca lcu la ted f rom t, heso, r(ssidues wh ich ,are ])rescnL in Lhe t ) ro te in in
l¢~ss Uhart |0 rt~aiduc-.~ t l)etcrt~h~cd a~ eysteic ~eid ~n(l cor rec ted f~)r ,(~0% rtwt)very ~fter per f i , rmh, ac id -ox idat ion
(Sc l~ra l~m, ~toore and l i igwo<)d, 19,~t).
I ' L . . ' I L I I , ' I ( ' ,X ' | ' |{ )N AX I~ ( : ( )M i~OSIT I (~N N ' l ' t ' l ) l l , ;N ()1 z f l~ .C l lYS ' I ' .~ , I , I , IN 263
A ~J~iJ,o-termimd ,mine ctcht deler~niuatim~ The fJ,-crystallin was converted into the. I)N t' derivnt ire t,)" t.lm me-t.bod of l~raewke, l -
(JortrnL, I'|arris and I,evy (1955). Two-(limer~sio, ull cltroll,~m)g,'al~hy (aec(irdin g t,o the met:l~,:~d of Levy, 1 954 ), of the et, her,sohlbh~" fraction of Lhe hydroly:ds mixt;urc revealed 2,4-dinit,.'oalfiline aml 2,4-dinitro|Jhenol only. The latter was verified by re.vm'sibh: decolorization with formic acid.
Chromnt:ogn'aphy of ~lm nqueous layer only rev(!a}ed ~-i)N[)-lysine and so/m: I)ack- gl 'O l~l td co lo l t r .
"I~AIII, E | 1.
Calculat.io~. ~f the appu.rcn~ s/)ec~fic volume of J,-erqsmllb~. from it.s omi~,'~ ncid composi- lion.
A tllint) n.ci~] res idue/ t 00 g
Amin, ) ac id re~/due ~,I pro6ein (wt°/~,) • ~ ¢× wt,',.},
Aspttrt,i[" m:id 4-73 0-60 2-838 Aspnrag inc ~ 4- 15 0"62 2,673 Th re, onine. 9_.5~ 0-70 - } .764 Ser inc 4- 8N 0-6:1 2,9,{ S (';lUt,~.ttli(" UOi([ ° 7"117 0-116 4.SI14 ('I t at La an i tic 6"4 7 0"t17 4- 3313 Proli,u: 3-22 0-76 2-447 G}yeine ,'b .qtl 0.64 2-543 A lau iae 3-45 0-%l 2"553 \ 'M ine ¢-07 0-86 3-500 ,Methinni,u; 3"(;.'~ 0-75 2-723 I s(~| et ll:ivt t~ 3-95 0"(10 :~-" 555 "L,etocine 5.5,t 0"1|0 4"986 ' l Jyrosine !)-2tl 0'7 l ¢5.575 ' t )Kenyhdanl ,t,~ 5".20 0.77 5-544 l<ysine .5.47 0"8:2 4-485 H is t id ine 4,-7!1 0-67 3-20~} A rgitxip.o 9- 99 0 .70 (i* 091{ ' l ' , 'ypto i f lmn 3-20 O" 74 2.36S Tota l lxMf-eyst ine 3-2 [ (I-03 2-022
Tota l 100- 16 72-825
* Ca lcu la ted f rom the re la t ionsh ip : wt (~g lu(aap)
res idua l n~qlee~dI,~r wMght glu(a~iJ) wtO/o_Nt l z ' N wtt'~°ghl wl~°~)asP ~: residue-/ utoJe¢mhLr woghg -- N I l ,
rosi(lm~t ~noleoular ~woight, g ' tu2r ' r - '~ i~u~'moleeu lav weight, a~ re.Mdua} molecu lar ,,','eight gl u NIuI ~{~'~i,,~ I [ ,)
The absence of mono- ~md di-DNP-histidine, and DNP-~rgitdne could be demon- s~ra|;ed by one-dimensiou~l ehrom~xtograp] W of the aqueous layer in the presence of di-DNP-histidine aud DNP-arginine as internal and lateral controls artd by the appli- co.tion of specific colour tests (P~uly- an({ Sakagucki rcacl;ion). A short hydrolysis (4 hr) of tile DNP-protehl with 11.2 N hydroeMoric acid in order to detect t, he labile DNP-prol ine, gave a negative result.
2fl.! A . ]". VAN DAS]
Finally, cystine or ~r half residne of cyst.ine, could be excluded as terminal amino groul~ after hydrolysis arid chromatogr~sphy of the DNI ' derivative of t~hc performic acid-oxidize.d protein.
These (L~ta lead to the conclusion that there is no free terminal amino group in fl,-crystaltin.
Carbo:mjl-terv~inal a,tino acid determination. ~.iNvo-dimensio,ml chromatography of the mixgure, obtained aftra' 16 hr of hydra-
zinolysation, yieMed glutamic acid as t.he most irnpor~tmt spot, but traces of tltreo~fine, serinc, glyeine and alanine were also t'mmd. The carboxyl end group was detcrmiimd in a qumltit~,.tive manner by enzymatic digest, ion with carboxypept~id:~sc A.
Products of the reac~ior~ at three time-intervals (~, 8 aad 20 hr) we,'e idc,tCified and esti,rmtcd by t reatment with FDNB. Sm'prisingly, very low yicIds of free. amino acids were obt~fincd.
GhtUm'dc ac.id appeared fi~t, but even at. a weight ratio (carboxypeptfdasc to fl,-crystaltin) of 1 : 25 a.ml even after 20 hr of digestion, ,m higher va.lue than 0-07 mole per mole of protein was reached. Traces of other amino acids, perhaps l)ro(htced by atttodigestion of t, he carboxypep~idase, were also fomld (0.009-0-04 moles). O,t ~t~e basis of these results it may stdl! 1)e concluded Omt ghttamie acid is the earboxyl- terminal re~sidu c of the proteirt.
The low amotmts, found in the quantfitative experiments, may be explained by asmtmit~g a proline residue iu the immediate neighbourhood of the terminal group. I t is knower that protine near to the C germinal end has tm inhibitory action on carboxy- i)eptidase A.
I t is noteworthy t;h.tt l~iekli, Ghazanfar, Gibbons and Edsall (1964.) made very sit,ti- ler obserw~,tions with carbonic ~mhydrases 5:ore h'mnan ery~chrocytes.
4. Discussion
The experiments described above show that fl,-crystMlin can be obtained by it combination of gel- and ion-exchange CtLromatog-ra.phy.
Th~ elec~rophoretic and altracentrifltgM afialyse.s indicate a high deg-ree of homo- geneity of the product. : 'he results of the amino acid a.mLlysis Icd to a molecular weighl; of 28,402 in agreement with the resuIts of the Archibald measurements.
This dat, um classes fl,-crystMlin with t, he low molecular weiglrt proteins,-confirming an earlier observation of .Rabaey (1959) from nlbrafil~ration ~tudies.
Based on the common assumption t-hat otfly molecula, r size. determine~s behav~ottr ht gel tilt, ration, it can be sga~d that 7-crystallins, fl~-, fl~-,/3 a- and pre-ec-ct3,st.aflin belong to the same eatx~go W a.s fi,-erystallim
t-[owcve.r, since-/~- ~md ~h-crystMlin emerge in ~hc rear l)a.rc of peak ":t (ct'. Fig, l(a.)), it is very likely thee the molecubu- weights of both proteius are slight.ly lower 5lamt that of ]?,-cryst,:filin. In agreement with this asstunption, ]3jgrk (1964) recently reported values between 19,000 and 20,01)0 for the moIecular weight- ofy-orystall ins, isoh~ted fl'om calf loltses.
Compar;,tive gel filtration of extract;s from ~alf-, cow-, pig- and horse-lenses (van Dam, manuscript it~ preparation) she/red that, the qua:at, itaLive prol:ortion of the compo~euts in the group of the low molcctflar weight proteins differs for the respective alfimals, a fact also noted t)y Rabaey (1965) with fishes.
PUI{IF ICATION ANI} COMPOSITION STUI)IE,S ()1" fl~.CRYSTAI, I, 1N 96,i
The f i i i lurc to find ~J.lty N - te rmi lml amino acid can b,: expMined by miltttoM/]g t.hat l lie termina l flllli.llO acid is blocked by lul t ict~tyl ~.rOllp, The l ) lorki l ig b3" li/~lcag(~ to ~t r~trbohydrat.r; gl'()up Ctlll t',o l.~xc.hldi.K.t by the recovi~ry of 100°/o in the amino aeld tl l l t l lys is.
1I; is notewort.hy tb_,lt. Bloentelidal, Bonl., Bene.d(st.l,i .'lml Wisse (19(15) inltde+ a simill~r observat ion wi~h highly ])urified ,~.-or.vatatlili. On f~lie oil ier hand, BjSrk (19(J4) fomict glycim~ as the only N-t~rmina l amino-acid in tit(; y-crystatllt~.s, isolat,.~l flx)m cattMenses, aad we fomzd g lutamic acid as N-terminal group in the major part. by far (if the fl-crystMlists ( iml)ut)l ished experiment.,;).
This emphasizes once moro i;tm eompl.exi~y of the [en8 prote'ins. A nalyse.~ of ea rboxyl- terminM amino acids of h.'.ns protcil is h:tvc noib ol'(.elt bc,zn l)crforined.
(.ilul:<~mic acid as carboxyl ~m-minal ,<l.mino acid ]ms not, yet bemi met witt, in any Ieris protein.. RceenrAy we tbund seriim as carboxvl ter ir l inM group +.ll" ~-cryai~ll l in (van Dam and ten Cat.e, 1.9(i6) and former ly we foruM t.|ireonilli2, i l l Sell'If) f3-eryst, allin.s (Vail D/till a-rid ~en Care, 1958).
Uiltbrt.m~at, ely, tile absence or blocking of tim N-tx;rminat amino arM, and the findilig of submotar ;tmolml;s ot ~ o~u-bo~Dd-termlnal grv, ips> make it, irnposaible rx~ answer t.he question whcf~her fl -crystal l in coimist~ of ore, or l;wo po ly l~pt ide chains.
]?rclimi~mry starch gel elcct.rophor(rsis ill 8. ,~ ~trea aim at p l t 3-0 did liot reveal more than oim band, suggesting t, he e.xistcrmc of ouly one polypcpt, ide chain. Itow(,ver, t.he possibi l i ty of S-S-bridge(s) between. 1;he c.hahts, if present, eannot~ hi., ruled ottt..
As for the occnrrence of fl,-orysttdlia, we timnd conll)onent~ in ext=.ract~s of calw.:~,~, horses at td pig le~mes with the same. electrophoret.ic mobi l i ty and occurring in the same gel filbrat;iotJ, f ract ion as bovil~c fi,-crys~,aliin,
l: lowevcr, immunoclcci;rophoresis of /3,-cryst.allin witl~ ant.i-chicken h.~rus m.ra did no~; reveal any arc, which might indicate t, lmt; t.his proi;ein does not occur in chick lens.
Coitoeridng the disvribut, i on ia the nd~tlt bovine lens, f l ;erysta l l ln wa.s fouttd both i~t the cortex and m tim lmdeus of $tte lens, but. the relat ive proport ion of f l , -crystal l ia ~o7:c altd y~-orysl;allins was much higher in the cortex than itl the mleleus. I:lowever, the total a mourie of tam low uiolecular weight: fraction was abotit 3 t imes greater in the nucleus t, han iu t;he cortex. In contraction witAl this, i t might; be worth wbih, ment ion ing tAl~tg Papaconstant, i~mu (1965) recettlJy fon.nd a difference in the eteet.ro- phorcbi.o mobilit, y of what he called t~ho <'),~erystaltin.¢' of ~>orts;x and mmteus. I t is t ikely thai; this is due to the. h igher ~tmounl~ of~q,-oryst.allit~ in the cortex.
AC, K N 0 %VLI'] l)O E~ {E N:I'8
The auVhqr is hxdcbted to Miss T. Opchmr, Mrs. M. G. d. vim Dam rsnd 3Ir~. M. (~P,11zejrI0.' i l for excellent technical assistance; to .i)r. D. 8t;hmhlt, Net!mrbmd.s institute for ])airy Re,search, ]~dc, for carrying out, bite ultracentrifugM analy.~e.~; to l)r. C. Mo~tIfoort, t,aborutory for Physiological Chemistry, St~lt~. >. Uuiver~ity, Utrecht, h>r i×-rf,Jrming the amino -ickt analyses li'nd the hydrazhlolyMs; to Prtifessor Dr. l[ . PA,>,'rn(~n,Ial ,Ltlrt Dr. ,l. W. .IJoot.sma for critical rein:iris% and l.o ~li-~. J. (I. J.lcidL, Mr. A. ,It. ,,'mi ll<~rs~-u and Slr. Th. ]~uiskens for ,skilled a,ssiatalicc in l)repllriilg till; llltlltti~c.rillb.,i,
! ~ E FER ]~N CES
Akn, bori, S., Ohns~, K. t~hd Narita. 14.. ([952~. t~ull. Chem. N<~.. Jap. 25, 21.I. :[~jiSrk, I. (196|). l~,xTll l"..'y, >. Hen. I, 145. BjSrk, 1. (19rJ4). E~.~tl Eye tle,~. 3, 16.
+'++'+ A+ F. VA.~ +>AM
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N~+ Y;++k, P,+ml;++Id P,+bti+hir~ + t"+.+m+~+++>', +1+~ i+ l,t+m++, P., Ter+..rkr+. C. +,+~d ,.f1.+l.++. +|. + tG++~l++ ~'.+.t/.+ri+..'~+*.+'+~ 20+ 204. ++|,+ira. l'. t t~+~), +I.'h,~+i~ t::,,,-++,+a.y ,,f t.~plmolm. |:°r~+,':t~++-l.('+,++:+t. l l . . Hmr+'~, ,J, I. ++M I++++",', A. 1++ (t~J~k+~). Jlt+,~,+l ++ ++..( Ithjcj++ni,++d +.| m,+l#x,.+. V,+L :~.
+'d. By t}. Gii0k+ N,.-w Vor~ |l'+tPl~<.i~-t+ry++,, }+. "+.~t), Pr+jlp>~, ,~. ~,J'~<i }++~t)+~+y, ,~+, +|t)+'L~+), .,+tat+ J . ())'.~ttlvr++pn,'.+l. ~+ .['+.., | | , "~-+'~. ]:r+++t+~'*'~,l~+ ,.)'. ~.~+i }~+Jh~+.?, ., ,~.~. !l ~++0). ,'Jr+.',/+. (.Ji+lJJ)~++r~.+>+. lCi*++~+++~.+ )` 6+. +)++}J. Frmaq, d++, +jl., I++ha++'. 31. ~,+t+'t Wh~me. J¢. (it+~l+.. l~mlt. ,%',¢... i%.~+~'. OpAl]u+l:+~d. 67. ~+;, Fr~.r~r,'+~i:,,, ,J,, RM~.m,+,y, M.. XV/;'+n:~, lt. ar+d Kami,.~ki+ +~l. (It~'+~l). +.l+iG. ,1. lff~l~+~l~+d. 42+ 577.
• fh~fm"+it, T.. W. m+d M¢+r~tt;, [*+. A. <. ItJ,l+;h lli~+h+.+++ ,.I..RL ++~).+~. i(+~+e.l+-/X+. 1. < I+~IO_L [+~++,+L ,<r(]+. P~,~I+~.., 82, .151. t~---,'~,. A. t.. ~lt l&t) . ;',,',+;mr," 174, i~t++. .~,|t+xz,+Jt,, W., An++r|rarh, "l °. |'. +~+~+j J|++tb<*r+, +~+ J'+ +l~+j~l), l'+.d,'ri+ti+++~, l 'r,~. 19, 3"++.i. +~+a~mk.i. W,. |l~.l|.~+r~t..~. 1'. +rj+l ,;~,~++++m~h+ T. P. ~}+36A++ ;,It(Z++ [l+++'J~+..+++. llh,l+.+.+.. 92, +12. ,~te~l+.,+~++~+ 3?. I... a++,+.+ 3h,+h+IL K, ¢1+i+,~? 3. ~+ri++++++ I t6 , t4:L .M+++r'~+t +.-" , <+.. T.. t+|+U4+. ~. l+~.y+~++d+ (.+/~e+e+. I~, i+l. P,~l+++<~+++.+++++.h++,~:~, ,~. <+ 111+-;,~+, ]+~,;~J+im,. l+h+lJ~+d+...,i+.-~+. I.(~+ ,~J. +++~+,,++++~ml+~+_il~.o+.+, .[.. ~:t+mt+Jk, ]:t+. +:L a++<) ;'~+I,.,. lq. (lg+++~L .l+i.+.+++,i~n. lli-.l++++, o.h'.~.~ GO, ~0,', |t~+:l. ]~, ;~. Jr+It] ~|OI/'+P+, ].. ~1+~'[~)+. ;++~+t+'fl.. ]~+]tllp+. ], ]~+~. )~.ml+~,+y, M. {.l ~J.+4U. "l'h+.+'6~ t '~J'.',,m+Ly of + ;1+*'+~1. FtM.,~+'y, M. +li+*.~+t i:'r~r+,/+x ,~ !1++ l+i~+¢++7ie,+l /"f~+id.+. Uo.lloq:zh.~zn []r+)+&++t, An;.~tot+latn. l+:l:,,,+.'i+:.: + +
l~+~h}m.}ti,+,~ C~.,ml++ny. H.ii,kti, +~. l+., ~ ;++~.z+.+f+.,r. ~..:X. N,, +.;d+t~++++. ]L |l: ~x~.++} I++Imti+ d. "I++ { }+,.~t+.i.++ J . l++'.,:d, i."l+e+~. ~+9, l+.'m.",.~= .~+h~+x+t}t.+ [L ++". t~t.+~d~-++i (+~t++, (.;. (|~+J}}. (++z.~, Ji+++:e+Pd+++J .~?,, ~~'J2+ .4++h.'mid+.. D. +;. +++d t++r: t",++<.. ~+, t;+t'a+~), ("h,-ra. i+:ee..~'~'d+~+l 58.: 2~3,
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