548 sn ORT COMMUNICA.TIONS VOL. 34 (1959)

B y employing this simplified method , sugar solutions to be analyzed by ion- exchange c h r o m a t o g r a p h y can be prepared in a short t ime, which is a great advan t age over the exist ing methods . Other advan tages a.re the smaller possibilities for the loss of sugars and hydrolysis of sucrose during the prepara t ion of sugar solutions. The chromatograph ic elut ion pa t t e rn of sugars ob ta ined by this me thod is, as far as the plant matt-rials we have thus far t es ted arc concerned, essential ly the same as those ob ta ined by the s t andard method.

We wish to express our sincere thanks to Prof. S. F U N A H A S H I , tile Univers i ty of Tokyo, for his interest in this work and to Prof. Z. NIKo~I, Osaka Universi ty , for his kind gift of s tachyose.

I~'IICHINOR1 N A K A M U R A

Departmcut o/Agric.ull~ral Chemistry, KENJI Morn the Uuiversity o/ Tokyo (Japa~)

z j . X . Ic, HY~ a.x:~ L , l ' . ZrI,r .I . A m . Chem. See . , 74 0 9 5 2 ) 2090- e G. t~. No(3c;t.~ AND L. P. Z|LL. Arch~ t t ioche'm. I l i o p h y s . , 4[ (1952) 21. a T. I:~'Kt:t a.~'l~ Z. NIKt:N1, . v i p p o n 3,*dgei-&'a¢'ukr* K u i s h i , in t h e p r e s s . 4 I£. ~I(~RI AND .'~i. NAIZAMUIIA, l]*~t], ,'Jg'r. ()]~¢~7~, .~oc. J a p a n . in ti~e p r e s s .

I t . G. t ' o x r t s . E . ( ' a m ~ hxz) L. F. I."CLOIEC, IHoch im, Bi*Jphys. ,4eta, 2e~ [1957) 146, .M. I)~;POTs, I'(. A. (~ILLI~.S, J~ I{. IIAx~II.TOX, 1', :X. REBERS AND |~. SMITH, :l ~zfl[. ()IICI1I., 2S (1050) 33o.

7 J , I t . ROl,:. j , tJi,,I. Chem. , xo7 {1934) 15. Received Decemher 23rd, z958

Changes in the ultraviolet absorption spectrum of trypsin associated with disruption of tert iary structure

Changes in the ul t raviolet absorpt ion spectra of several prote ins have heen demon- s t r a t ed when these were exposed to a var ie ty of exper imenta l condit ions. "File changes may arise from the ionization of the tyrosine h y d r o x y l gr0upsX,2, a or from the dis- rupt ion of hydrogen bonds involving tyros ine h y d r o x y l groups% a. I t has also been suggested tha t a change in charge of an ionizing group neighbour ing a non-iontzable chromophor ic group (e.g. t r yp t ophan ) m a y result in a shift in the absorpt ion spec t rum of the la t ter to lower wavelengths ~. Recent ly , Cm~.RV~XK,\ has shown tha t the act i- va t ion of chymot ryps inogen is accompanied hy a shift in the absorptioxl of the tyros ine residues to lo~er wavelengths 7. He also demons t r a t ed a spect ra l change accompanying the autolysis of c h y m o t r y p s i n similar to tha t which occurred during urea t r e a tme n t of e i ther the zymogen or the enzyme. Under bo th condi t ions of de- na tu ra t ion , the difference spectrunl resulted from a wavelength shift in the at)sorption of t rypLophan and prol ,ably of all the ehromophor ie amino acid residues.

We have been una.l;)e to d,~.monstrate a significant change in absorp t ion dur ing the act ivat ion of t rypsinogen, bu*. have orJ.~ervcd a character is t ic and reproducible diiieJence spec t rum when t ryps in was au to lysed in the absence of calcium or upon t r e a t m e n t with urea. These spectra Mlow a s tr iking s imilar i ty to those ob ta ined by Ct~]~v]:xK,x with the chymot ryps i n system.

In all exper iments at pH &oo, the reference cuve t t e of a Beckman D K - I re-

VOL. 3 4 ( I 9 5 9 ) aIIr)lgT COM~fUNIC.~TIO.XS 549

cord ing s p e c t r o p h o t o m e t e r c o n t a i n e d ~.8I mg t r y p s i n / m l in o.~ .'ll t r i s ( h y d r o x y - m e t h y l ) a m i n o m e t h a n e , 0.05 .'~I CaCI.,. The test cuvet~e c o n t a i n e d 3.5 mI of the e x p e ~ - m e n t a l so lu t ion and, at zert) t ime , o .r ml of 6.50 % tryps in so lu t ion wa~s added w i t h a rapid m i x i n g dev ice dcsi~,ned by G. H. D i x o n of this l a b o r a t o r y . . M e a s u r e m e n t s of the a b s o r p t i o n difference at 293 m p were b e g v n wi th in z o sec and difference spec tra f rom 330 m p to z 4 o m p were takett at su i table intervals . T h e t e m p e r a t u r e w a s m a i n - ta ined at e5 ~k o .~ ° t h r o u g h o u t .

The decrease in a b s o r p t i o n of tryps in at z93 m / , in o .z M t r i s ( h y d r o x y m e t h y l ) - a m i n o m e t h a n e , p H 8 .oo in the absence of ca l c ium is s h o w n in Fig. z t oge ther w i th a s imi lar e x p e r i m e n t in the presence of o .oz M- CaCI~. Since the a u t o l y s i s o [ t ryps in in the absence of ca l c ium has been a m p l y d e m o n s t r a t e d a,", ther'.: s e e m s l itt le d o u b t t h a t th i s process is a c c o m p a n i e d by a character is t ic spectral change (Fig. ~).

T h e ef fects of 2 3 f and 8 . lI urea at pH 8 .0o on the absorpt ion of t ryps in at ~93 ml~ are s h o w n in Fig. 3. In the absence of ca l c ium (Fig. 3A), the spectral change

4.0. ,?

3.0,

/ - /

o 4'0 8o ' ~ o | ~ o ' MINUTES

Fig . ~.

a6o

:il// V a4o E:60 ~ 8 0 3C)0 3~O

WAVELENGTH IN m/,~ F i g . a-

0 10 ~'0 3 0 4 0 BO ~ 0 70 M!NUT.'-~,

F t g . 3-

F ig . 1. C h a n g e hi abs<n ption at -'07, m I t of t r y p s i n .d~r ing a ~ i t ~ l v s i s : l t 3 3 : : " x t ~ ' p s i n i n ¢ , . I . l / t r i s ( h y d r o x y - n l e t h ~ ' l ) a n l i n l ) m c t h a n e , p H 8.o(>. n o c a l c i u m : • r - -

t r y p s i n hi o.t)) .1[ ( ' aCid , o . I . l l t r i s t h v d r o x y m e t h y l ) - an l i t l t ) l l le~l l3ne , p H I~1.00,

FiR. 2. I) i f ference sp e c tr a of t r y p s i n sn lut ions at - ' 5 : ; - - a f t e r : 7 o - m i n s u l t o h ' s i s in o.~ .1/ t r i s ( h v d r o x y - i~lethyl)aillilLon|l:thi~n¢~, p H 8~oo, no ca l¢ iu r~ ; . . . . . after 5 rain in S .11 u r e a . o.z . l [ t r i s (h3"d roxy~r t ezh .v l ) an l ino -

m e t h a n e , p H S.oo.

Kl~4. 3- C h a n g e s in al)s~jrption at 203 nl/~ of t ryps in in ~ .'~[ ( ' , : . ".:) an t i z 3 / ( g O1 u r e a . . k . T r v p s i n i n u r e a , o . i .i[ t r i s ( h v d r o x y m e t h y l ) a r , l i n o m e t h a n e , p i t 8.00, no c a l c i u m . Is. T r y p s i n in urea, o. I . l l t r i s~hydrc~ .~ymetby |} - a m i m > : n c t t m n e , o "5 .11 {"aCl~. p H 8 .o0 . C. Tryps in in

urea, p t I 3.00.

55o SNORT cox~V~lC, t r to~s voL. 34 (1959)

is ins tantaneous in 8 M urea, but proceeds at measureable rates a t lower concen- trat ions. The presence of calcium (Fig. 313) marked ly reduces the rate of the spectral change. Similar effects of calcium on" the chymot ryps in system were observed by C~nsvr. r~K:xL The spectral changes at pH 8.00 in solutions of urea less concentra ted than 8 M probably result from both the effects of urea and of autolysis since t ryps in is k n o ~ to be autolysed at lower concentrat ions of urea even in the presence of calcium '° .

In artother series of experiments the effects of urea on t rypsin at pH 3.oo were followed (Fig. 3C). In these exper iments the reference cell of the spect rophotometer contained x.8~ mg t ryps in /ml in o.ooi M HCI. It is of interest t h a t a t each urea concentrat ion the rates were g rea te r ' than at pH 8.o0 in the absence of calcium.

The effects of several acidic solutions on the absorpt ion spect rum of t ryps in were s tudied with t rypsin at pH 8.oo as the reference. The changes in molar ext inct ion at 293 mg in solutions of pH 5.o, 3,o, and x,o were o,r, 0.8° and 1.3, respectively. The.-;c changes were ins tan taneous and at pH 3.o were not affected by the presence of calcium. The da ta are inadequate to ascribe the changes to a dependence on the ionization-, of carboxyl groups as has been clone for lysozymc ~.

In a prel iminary exper iment the difference spect rum of t rypsinogen in 7-5 M urea was found to be similar to t h a t of t rypsin in urea. These spectral changes do not involve the active center of t rypsin, since no change in absorbaney was observed during the act ivat ion of t rypsinogen or during reactton of t ryps in wi th diisopropyl- f luorophosphate. Fur the r studies on the factors affecting the rates and magni tudes of these spectral changes are contemplated.

This work has been supported in par t by a grant of the National Ins t i tu tes of Hea l th (RG-4617) to Dr. HASS NEURATH whose interest and advice th roughou t this work is grateful ly ack,.~owledged.

L A W RE N CE B . SMILLIE*

Depart.men.t o] I-Jiochemislry, (..'~dvcrsity o/ H"ashi.~gton, Seattle, l~ ash. (U .S .A . )

1 l), SHUG-XR. l¢iochem, j . , 52 (1952) 14 a. ('. T.:~N~:ORD, J. l~. Hat,~.~sT~x AXD D. G. R,'~Js, J. :Ira. chet,,..'~oc., 77 (19.55) cqo9.

:/ | ' . E. ~:ILCO?-', Fcdcrati~r Prec., I6 f i957) 270, I t-I. A. %CHt,:R~nA. Btochtm. Biophys. ,-Iota, z3 f1957) 1¢j6. r, M. I.AgKOWSK|, JR., J . .~I . \VII)O.',I, .'ll. L. }IOt:AI)I)l~-X AND l - I . . \ . SCHF.IIAGA, #~iOC#~im. lJioflhJ,'s.

..|¢l,t, 1~ (1956} 581. j . W. ])o-;:~,'.ax, M. I,.~sK,ws~cl, JR. ,~.~D t t . A, ~CHb~;d.~,GA, 13iochim. 13iophys...1eta..:~ (t~)58 } 455-

7 C. i r, ('HER']E.NKA, ~iOCh~$Yl. l~ioph~'S..'tfla, 3t (19.59! 85- 8 l,, (;OIJ-INI, tllo~:hi~Jl. IJiophys..4~t¢t, 7 (19511 3 I8. 9 [.. "k~,-. CUNNINGItASa, JR. , ]-:. Tl PL'~'ZE, N. .~ i . GREEN &Nil |1. NIr. URATIt, I)iStZfSSiOJlS Fat'ada), See.,

I3 (t93- ') 58, l~b T. \ ' iSWANA'IHA AND I. E . LI!~-NIr-R, J . 13iol. Glwm., 215 (1955) 777-

Received December Io th , x958

" I)c;nner l"elhJ~k of Medical Resea rch e l t he l I iv i s ion of Medical Science, Na t iona l A c a d e m y ~t S u i c n c c s - . Na t i ona l I~cs2areh Counci l , ~957-58, (m Leave of ab s en ce f r o m the D e p a r t m e n t of l h o c h e m i s t r y , U n i v e r s i t y of AI l~ r t a , l ' : d m o n t o n , Albe r t a , C a n a d a .