BIOCHIMIE, 1971, 53, 1085-1093.

Polarimetric and chromatographic investigation of' the transfbrmation of $-lactoglobulin A and B

upon alkaline denaturation.

irreversible

H a r r y ROELS (*), Gisble PR~AUX and Ren6 LONTIE.

L a b o r a t o r i u m voor B i o c h i m i e , Ka lho l i eke Univers i te i t te Leal~en, Dekens t raa t 6, B-3000, Louva in , Be lg ium.

(18/10,/71).

S~lmmary. - - The immedia~.e and reversible N ~ ± - ) ~ R conformational t ransi t ion of ~-lactoglobulin A and B, occurring from pH 6.8 to 9.25 at 20 ° , is characterized by a t ransformat ion of 15 p. 100 B-structure into random structure, while the a-hel ix content remains constant.

Above pH 7.4 the laevorotat ion of [3-A and ~-B solutions, saturated with air, increases as a function of time, the faster the higher the pH. This alkaline denaturat ion causes an irreversible t ransformat ion of [3-structure to random structure, whi le the a-helix content hardly changes.

At pH 8.9 and 20 ° the irreversible conformational change is accompanied by the format ion of at least two groups of components eluting faster on Sephadex G-100 than unal tered ~-lactoglobulin. These components arc insoluble at pH 5.2. A marked decrease in thiol content is also observed.

The increase in ]aevorotation, the polymerization, and the decrease in thiol content due 1o alkal ine denaturat ion are near ly twice as fast at pH 8.9 and 20 ° for ~-A than for ~-B.

I N T R O D U C T I O N .

~ -Lac tog lobu l in has a m o l e c u l a r w e i g h t of 36 000 in the i s o e l e c t r i c r eg ion (pH 5.2) [17. It cons is t s of t w o i d e n t i c a l p o l y p e p t i d e c h a i n s con- t a i n i n g one t h io l g roup and two d i su l f ide b r i dges each . T h e d i s s o c i a t i o n in to ha lves [2, 3] i n c r e a s e s f r o m p H 5.5 to 8.5, n e a r pH 7.5 a r e v e r s i b l e con- f o r m a t i o n a l t r a n s i t i o n ( N ~ : ~" R) occurs , a c c o m - p a n i e d by an i m p o r t a n t i n c r e a s e in l a c v o r o t a t i o n and by the e x p o s u r e and ion i za t i on of t w o h i d d e n c a r b o x y l g r o u p s [4, 5, 61. Above p H 7.5 th is fast N ~ R t r a n s i t i o n is f o l l o w e d by a s l o w e r i r r e v e r s i b l e c h a n g e (a lka l ine d e n a t u r a t i o n ) [7, 8]. Such a two-s t ep m e c h a n i s m was also found for the u rea [18, 9] and hea t [10, 111 d e n a t u r a t i o n of ~- lac toglobul in .

The o l d e r l i t e r a t u r e on the d e n a t u r a t i o n of ~- lac toglobul in dea ls w i t h p o o l e d o r no t spec i f ied gene t ic va r i an t s . T h e s e q u e n c e of ,~-A d i f fe r s f r o m tha t of ,~-B by the subs t i tu t ion in e a c h p o l y p e p t i d e c h a i n of Asp a n d Ala for Gly and Val r e s p e c t i - ve ly [12, 13]. T h e s e d i f fe rences , a l t h o u g h small ,

(*) Graduate fel low of the Insti tuut tot Aanmoedi- ging van het Wetenschappel i jk Onderzoek in Nij- verheid en Landbouw.

Abbreviations : ~-A : ~-lactoglobulin A, ~-B : ~-lactoglobulin B,

PMB : p-mercuribenzoate.

i n f luence seve ra l p r o p e r t i e s [14, 115, 10, 17J of these gene t i c v a r i a n t s and a m o n g t h e m the i r r e l a t ive r e s i s t a n c e to t h e r m o d e n a t u r a t i o n . In this respec t , at 70 ° a n d p H 6.7 I~-B a p p e a r e d m o r e suscep t ib le to h e a t t r e a t m e n t than ~-A Ell, 18], and .8-C [19] even m o r e than ~-B. DUPONT [ l l ] e m p h a s i z e d that the d i f f e r e n c e in d e n a t u r a t i o n ra te b e t w e e n ~-A and ~-B was due to the s low i r r e v e r s i b l e step.

The p r e s e n t p a p e r dea ls w i t h a c o m p a r a t i v e s tudy of bo th the r e v e r s i b l e and i r r e v e r s i b l e i n c r e a s e in l a e v o r o t a t i o n found fo r ~-A and .B-B at 20 ° as a f u n c t i o n of pH. T h e r e v e r s i b l e step was s t u d i e d by m e a s u r i n g the o p t i c a l ro ta t ion in the v i s i b l e and n e a r u l t r av io l e t 39 m i n a f te r solu- b i l i z a t i on of the p ro te ins , and the i r r e v e r s i b l e one by f o l l o w i n g the i n c r e a s e in l a e v o r o t a t i o n as a f u n c t i o n of t i m e and by c o m p a r i n g the ra te cons tan ts . In mos t cases the c o n t e n t of a-hel ix , ~- and u - s t ruc tu r e ( u n o r d e r e d ) was ca lcu la ted . The r e v e r s i b i l i t y of the i n c r e a s e in l a evo ra t a t i on of ~ - l ac tog lobu l in A and B kep t at pH 8.9 and 20 ° was c h e c k e d by l o w e r i n g the pH. Also at tha t pH the s e c o n d a r y t r a n s f o r m a t i o n s r e s p o n s i b l e for the i r r e v e r s i b l e step w e r e i n v e s t i g a t e d by gel fi l tra- t ion on S e p h a d e x G-100, by u l t r a c e n t r i f u g a t i o n , th io l d e t e r m i n a t i o n , so lub i l i ty m e a s u r e m e n t s at pH 5.2, and p e r f o r m i c ac id o x i d a t i o n .

Some resu l t s of th is p a p e r w e r e r e p o r t e d in a p r e l i m i n a r y no te [20]. A c o n t r i b u t i o n to the effect

1086 H a r r y Roe l s , Gis~;le P r g a u x a n d R e n 6 L o n t i e .

of pH on ~-lactoglobulin A, B and C was in the meant ime given by McKENZIE and SAWYER [21].

MATERIALS AND METHODS.

Prote in solutions.

~-Lactoglobulin A and B were prepared f rom milk of typed cows [22] accord ing to the method of ASCHAFFENBURG a n d DREXVRY [23]. The dialyzed pro te ins were lyophi l ized and stored at 4 °.

Prote in solutions of about 20 g/1 were prepared in the fol lowing buffers (I 0.1) : sodium acetate- acet ic acid (pH 5.0-5.6), NaeHPO4-KHePO ~ (pH 5.6- 7.2), and Tris-HC1 (pH 7.2-9.25). The protein con- cent ra t ion was de te rmined wi th a Beckman DU spec t rophotomete r at 280 nm using A (1 p. 100, 1 cm, 280 rim) = 10.0.

The pH values were measured with a Beckman Model G pH-meter, glass e lec t rode 290, and in the revers ib i l i ty and solubil i ty exper iments wi th a Beckman Expanded Scale pH-meter Model 76, glass e lectrode 39167 D-4.

Thio l de terminat ion .

Thiol de terminat ions w e r e car r ied out in Tris- HC1 buffer (pH 8.2, 1 0.1) accord ing to the method of BoY~R [24] (reaction time, 16 hours), p-Mercn- r ibenzoate (PMB) (Laboratory Reagent, BDH, Poole, England) was puri f ied as descr ibed [24], a 0.01 M stock solution was prepared as f o l l o w s : 35.7 mg purif ied PMB were solubil ized in 3 ml NaOH 0.1 M and 3 ml bidis t i l led water . The pH was adjusted to 8 wi th HC1 0.1 M ; the end volume was brought to 10 ml wi th bidis t i l led water. The concent ra t ion was checked spec t rophotometr i - cal ly at 232 nm in phosphate buffer (pH 7.0, I 0.1) us ing e M : 1.69 X 1041. mol 'L cm -1.

P e r f o r m i c acid ox idat ion .

Per fo rmic acid oxidat ion of native and alkal ine dena tured ~-A and ~-B was car r ied out accord ing to the method of MOORE [25], slightly modified. Instead of the HBr t reatment , that yielded pro- ducts insoluble at pH 8.2, a rapid dialysis against Tris-HC1 buffer (pH 8.2, I 0.05) at room tempe- ra ture was used to remove the excess of pe r fo rmic acid. After 4 hours the dia lyzed solution became viscous, but the viscosi ty dropped again after three renewals of the d ia lyzing buffer. After dialysis against b idis t i l led wate r the oxidized products were lyophi l ized and stored at 4 °.

Optical rotation.

Optical rotat ion measurements were per formed at 365, 405, 436, 546, and 578 nm at 20 ° wi th a m.anual Zeiss photoe lec t r ic po la r imete r LEP (pre-

cision 0.005 °) (Oberkochen, West-Germany) on prote in solutions (20 g/l) in thermostated cuveltes of 10 cm light path. The tempera ture of tile solu- tion in the cuvettes was control led wi th a ther- mocouple TE-3 (Ellab, Copenhagen, Denmark) .

The reduced mean residue rotation [m'] 7, was calculated from :

3 M R W [m'] 7. = • [tt] 7, (1)

n 2 + 2 100

The mean residue weight , M R W , for the ~-]acto- globulins was taken as 112 [26]. The Lorentz correc t ion factor, 3/(n2 -4- 2), of the buffers was calculated from the ref rac t ive index measured at 20 ° wi th a Pu l f r i ch cr i t ical angle refracto- meter (Bellingham and Stanley, London, England) at 436, 546, and 578 nm. The re f rac t ive index at 405 and 365 nm was obtained by extrapo- lation. -- tot] ), refers to the specific laevorotat ion. In one case the r educed specific ~aevorotation --rrt '~ ), was also considered. It cor responds to

3 - - [ ~ 1 7, n 9 + 2

The rotatory d ispers ion parameters , a,, and b o, were computed by the method of the least squares from the MOFHTT-YANa equat ion [27] :

,o (2) tin'l), : ao : ~ ~ + bo 7, A - - l , o

where ?'o was taken as 212 nm [26].

The percentage of each structure was calculated f rom :

a o = ~(ao)~ + f3(a o) ~ + U(ao) . (3) bo = ¢t(bo)a-4-,~(bo) ~ -4- U(bo) u (4)

ct + ~ + u = 1 (5)

where a, ~ and u re fe r to the fract ion of the pro- tein molecule in the a-helical , 8- (pleated sheet) and unordered structure. These s t ructures are character ized b y : (ao)~ = O, (a o) ~ = + 400, (a,)a . . . . . 650 and (b¢)~ = - - 630, (bo)~ - 0, (b,,)~ = 0 [28].

Revers ib i l i t y exper imen t s . ~-Lactoglobulin A and B, kept at 20 ° in Tris-

HC1 buffer (pH 8.9, I 0.1), were invest igated as a funct ion of decreas ing pH. F rom a solution (22 g/l) 10 ml samples were taken at given t imes and the pH progress ive ly lowered to values between 8.8 and 6 by adding HC1 4 M slowly wi th an Agla m i c r o m e t e r syringe (Burroughs Wellcome, London, England). The laevorota t ion of the adjusted solutions was measured after 1 hour.

Gel f i l trat ion. Sephadex G-100 (Pharnmcia, Uppsala, Sweden)

was swollen for at least 48 hours in Tris-ItCl

BIOCHIMIE, 1971, 53, n ° 10.

A l k a l i n e d e n a t u r a t i o n of Glac tog lobul in A and B. 1087

buf fer (pH 8.2, 1 0.05). The c o l u m n (70 cm X

3.2 cm) w a s m o u n t e d and filled as d e s c r i b e d E29]. The p a c k e d c o l u m n w a s p e r c o l a t e d w i t h the buf fe r for t w o days . The o p e r a t i n g p r e s s u r e n e v e r e x c e e d e d 25 cm. T h e e lu t ion was p e r f o r m e d at r o o m t emppera . tu re at a f low rate of 20-30 m 1/hour . The e luate w a s co l l ec t ed in 2 ml f r a c t i o n s and the p ro t e in usua l ly loca ted by m e a s u r i n g the absor- bance at 280 rim.

The c o l u m n w a s ca l i b r a t ed w i t h a m i x t u r e of 40 mg a - h a e m o c y a n i n of Helix pomatia ( tenths at pH 8.1, m o l e c u l a r w e i g h t 990 000) [301, 60 mg d i s u l f i d e - d i m e r of b o v i n e s e r u m a l b u m i n (molecu- la r w e i g h t 138 000), 60 m g m o n o m e r o f b o v i n e s e r u m a l b u m i n ( m o l e c u l a r w e i g h t 09 000), 70 mg ~- lac toglobul in A o r B (d i s soc ia t ion e q u i l i b r i u m at pH 8.2) [2, 3], and 50 mg m y o g l o b i n ( m o l e c u l a r w e i g h t 10 000) o r c y t o e h r o m e c ( m o l e c u l a r w e i g h t 13 000) so lub i l i zed in 3 ml of the e lu t ion buffer . An e q u i v a l e n t h e i g h t of one t h e o r e t i c a l p la te of 0.9 m m was usua l ly ob ta ined .

Ultracentrifugation.

The s e d i m e n t a t i o n ve loc i ty e x p e r i m e n t s w e r e c a r r i e d out at 20 ° in a Sp inco Model E ana ly t i ca l

u l t r a c e n t r i f u g e e q u i p p e d w i t h t he s e h l i e r e n opti- cal sy s t em and a t e m p e r a t u r e c o n t r o l un i t (Beck- man I n s t r u m e n t s , Inc. , Pa lo Alto, Calif . USA). The s e d i m e n t a t i o n s w e r e p e r f o r m e d at 59 780 r e v . / m i n in an a n a l y t i c a l s ingle s e c t o r cel l (12 ram, 4 °) on samples (9 g/ l ) so lub i l i zed in Tris-HC1 buffer (pH 8.2, I 0.1, NaN 8 0.08 p. 100). The s ed imen ta - t ion p a t t e r n w a s eva lua ted by m e a n s of a Nikon Prof i le P r o j e c t o r Model 6C ( N i p p o n Kogaku K.K., Tokyo , J a p a n ) .

Solubili ty experiments.

F r o m a so lu t ion of G l a c t o g l o b u l i n A and B (20 g / l ) in Tris-HC1 buf fer (pH 8.9, I 0.1), s amples of 4 ml w e r e p e r i o d i c a l l y taken . T h e i r p i t was l o w e r e d to 5.2 by a d d i n g s lowly a def in i te v o l u m e of HC1 4 M w i t h an Agla sy r inge . A p r e c i p i t a t e o c c u r r e d in all the ad jus t ed samples , e x c e p t in those t aken a f t e r 30 min . Af ter c e n t r i f u g a t i o n for 20 rain at 4 500 r e v . / m i n , exac t l y 3.5 ml of the s u p e r n a t a n t , ad jus ted to p H 4.7, w e r e e h r o m a t o - g r a p h e d on a S e p h a d e x G-100 c o l u m n (70 cm × 3.3 cm) in t r i e t h y l a m m o n i u m a c e t a t e - a c e t i c ac id buf fe r (pH 4.7, I 0.05) a c c o r d i n g to the p r o c e d u r e d e s c r i b e d for the gel f i l t r a t ion on S e p h a d e x G-100 in Tris-HC1 buffer (pH 8.2, I 0.05).

7o "%so

b 70 - 0 5 0

IK3 -[~]

- [~ 9o

7 0

5 0

4

,.,)y/J .., .o'.IY/2/ ° .~.,~," .J," pP

6 7 8 pH 9

10(3

60

33O

290 ~ 50

- a o

4 0

250

3C 210

170

130

2C

IC

J B

h A

Fro. 1A. - - Reduced specific laevorotat ion - - [ a ' ] ( . . . . ) and reduced mean residue rotat ion - - [ m ' ! ( - - ) at 436 nm, and rota- tory dispersion parameters --ao ( --.--.-- ) and --bo as a function of p H ; 8-1actoglobulin A (open symbols) and B (closed symbols). The measurements were performed at 20 °, 30 min after solubilization of the proteins in buffers of 1 0.1.

Fro. lB. - - Percentage of a-helix ( ~and • ), 8-structure ( /2and • ), and unordered structure ( O and • ) of 8-1actoglobulin A (open symbols) and B (closed symbols) as a function of pH. The calculations were based on the ao-- and bo-- values of Fig. 1A.

BIOCHIMIE, 1971, 53, n ° 10.

1088 Harry Roels, Gis~le Prdaux and Rend Lontie.

R E S U L T S .

Optical rotation of ~-lactoglobulin A and B as a function of pH.

T h e op t i c a l ro t a t ion of B-A and B-B w a s measu- r e d in the p H - r a n g e 5 to 9.25 at the five wave - l eng ths , 30 m i n af te r so lub i l i za t ion . A s l ight l i n e a r i n c r e a s e of the r e d u c e d spec i f ic l aevoro - t a t i on and of the r e d u c e d m e a n r e s i d u e ro t a t ion b e t w e e n p H 5 and 6.8 w a s f o l l o w e d by a m o r e i m p o r t a n t i n c r e a s e f r o m p H 6.8 to 9.25, as i l l u s t r a t ed in Fig. 1A for the m e a s u r e m e n t s at 436 nm. The va lues fo r f3-B w e r e s l ight ly m o r e n e g a t i v e than fo r ,~-A.

T h e Moffitt p lo ts at the d i f f e r en t p H va lues w e r e l inea r , the va lues of ao and b o had an a v e r a g e c o r r e l a t i o n coe f f i c i en t of 0.9981 _ 0.0022. By i n c r e a s i n g the pH f r o m 5 to 9.25 the b o va lue of I%A and ~-B c h a n g e d on ly s l igh t ly and l i nea r ly , f r o m - - 6 1 . 4 ° t o - - 6 8 . 1 ° and f r o m - - 6 0 . 8 ° to - - 6 8 . 2 ° r e s p e c t i v e l y (Fig. 1A). On the c o n t r a r y a o v a r i e d c o n s i d e r a b l y w i t h p H in a w a y s im i l a r t o - - [ a ' ] a n d - - [ m ' ] .

1001

70

6O

"6 2

a n

°t6 n6o 2bo 3bo

S a

- ± _= ._ ,~ •

6 ~6o 2bo 3bo ' hours

Fie. 2. - - Percentage of (t-helix (A and • ), B-struc- ture (0 and t ) , and unordered structure ( © and • ) of ~-lactoglobulin A (open symbols) and B (closed symbols) as a function of time. The proteins were kept at 20 ° in Tris-HC1 buffers, I 0.1, p H : (a) 7.41, (b) 8.01, (c) 8.21, (d) 8.64, (e) 8.83, and (f) 9.25.

T h e p e r c e n t a g e s of a -he l ix , B- and u - s t ruc tu re w e r e ca l cu l a t ed for B-A and (5-B for each p H va lue (Fig. 1B). In con t r a s t to the r a t h e r cons t an t a -he l ix c o n t e n t of 10.2 p. 100, the .B-structure d e c r e a s e d for I~-A and B-B by about 15 p. 100, w h i l e the u - s t ruc tu re i n c r e a s e d by n e a r l y the

same amoun t . T h e ,~-structure con t en t w a s s l igh t ly h i g h e r for B-A t h a n fo r ~-B, the u - s t ruc tu re c o n t e n t s l igh t ly l ower . T h e s e op t i c a l r o t a t o r y d i s p e r s i o n da ta a re cons i s t en t w i t h the l i t e r a tu re [4, 5, 6, 26, 28, 31, 32].

Optical rotation of ~B-lactoglobulin A and B at alkaline pH as a function of time.

The l a e v o r o t a t i o n w a s m e a s u r e d at the five w a v e l e n g t h s on so lu t ions kep t at 20 ° in Tris-HC1 buffers , I O.1, s a t u r a t e d w i t h a i r . T h e spec i f i c laevoro~tations at 436 n m as a f u n c t i o n of t i m e h a v e been p u b l i s h e d [163. At p H 7.41 no c h a n g e w a s obse rved . Up to p H 8.64 the spec i f ic l a evo ro - ta t ion i nc r ea se w a s smal l and v e r y s low, but f r o m p H 8.64 to 9.25 it b e c a m e faster . ,~-A a l r e a d y r e a c h e d a final --[a]43620°=m va lue of 176 ° a f t e r 384 h o u r s at pH 8.64, w h e r e a s $-B did not, even at pH 9.25.

. ~10 o

x

8 9 7 8 pH 9

FI~. 3. - - Firs t-order rate constants of the change in specific laevorotat ion at 436 n m ( A and &) , in B-structure ([] and • ), and in unordered structure ( © and • ) of ~-lactoglobulin A (open symbols) and B (closed symbols) as a function of pH. The proteins were kept at 20 ° in Tris-HC1 buffers (pH 7.41 - - 9.25, I 0.1).

The Moffitt p lo ts of the r e d u c e d m e a n r e s i d u e ro t a t ion y i e lded s t r a igh t l ines. F r o m the r o t a t o r y d i s p e r s i o n p a r a m e t e r s a o and b o, the p e r c e n t a g e s of the th ree m a i n s t r u c t u r e s in p r o t e i n c o n f o r m a - t ion w e r e ca l cu l a t ed (Fig. 2). Over the c o n s i d e r e d pH range the a -he l ix con t en t of B-A and B-B d id not s h o w any s ign i f i can t change w i t h t i m e as i l l u s t r a t ed by one e x a m p l e for B-A and $-B at p H 9 .25 ; the B-st ructure , h o w e v e r , d e c r e a s e d w h i l e the u - s t ruc tu re i nc r ea sed .

BIOCHIMIE, 1971, 53, n ° 10.

Alkal ine denaturat ion of ~-lactoglobulin A and B. 1089

Kinetics of the alkaline denaturation of ~-A and ,~-B.

The change in spec i f i c l a e v o r o t a t i o n , - [u]~°¢'6 ~ . and in ~- and u - s t ruc tu re d u r i n g the a lka l ine d e n a t u r a t i o n of ~-A and ~-B at s eve ra l p H va lues obeyed f i r s t -o rde r k ine t i c s , by a p p l i c a t i o n of the in t eg ra l m e t h o d [33]. The ra te cons tan t s , ca lcu- la ted by the m e t h o d of the least squa res , a re r e p r e s e n t e d in Fig . 3 as a f u n c t i o n of pH. T h e y s h o w e d tha t ,~-A w a s at least t w i c e as su scep t i b l e to a lka l ine d e n a t u r a t i o n above pH 8.6 as ~-B.

170

150

130

I10

9 0

7C 7 8 9 7 8 p H 9

in the so lu t ion . T h i s was c o n c l u d e d by c o m p a r i n g the spec i f i c l a e v o r o t a t i o n of 8-1actoglobul in A and B f o u n d u p o n l o w e r i n g the p H to 8.8, 7.5, a n d 6.4, w i t h tha t f ound also at those p H va lues fo r both p r o t e i n s k e p t fo r 1 h o u r and 384 h o u r s in Tris- HC1 buf fe r (pH 8.9, I 0.1) at 20 °.

Transformation of ~-A and i~-B accompanying the increase in laevorotation during alkaline de- naturation.

Gel filtration on Sephadex G-IO0 at pH 8.~. Solu t ions of ~-A (20.6 g / l ) and of ~-B (21.6 g / l ) in Tris-HC1 buf fe r (pH 8.9, I 0.1) w e r e kep t at 20 °. Samples of exac t ly 3.2 ml w e r e t a k e n af te r g iven t imes a n d c h r o m a t o g r a p h e d on a S e p h a d e x G-10O c o l u m n in Tris-HC1 buf fe r (pH 8.2, I 0.05) (Fig. 5). A lka l ine d e n a t u r e d ~-A and ~-B r e m a i n e d comple - te ly so lub le in the e lu t ion buf fe r as c o n t r o l l e d by d ia lys is .

FI6. 4. - - Reversibi l i ty of the specific laevorotat ion at 436 nm and 20 ° of fl-lactoglobulin A (©) and B ( e ) , kept f o r : (a) 1 hour, (b) 24, (c) 72, (d) 168, (e) 264, and (f) 384 hours at 20 ° in Tris-HC1 buffer (pH 8.9, I 0.1), by lowering the pH of the samples progressively with HC1 4 M. Dashed line : specific laevorotat ion of ~-lactoglobulin A and B measured 30 rain af ter solubi- l ization at the given pH values.

E~ u ¢

Reversibility of the laevorotation o[ alkaline denatured ~-A and [~-B.

Fig. 4 r e p r e s e n t s as a f u n c t i o n of p H the r e v e r - s ib i l i ty of the spec i f i c l a e v o r o t a t i o n of ~-A and ~-B, kep t at 20 ° in Tris-HC1 buf fer (pH 8.9, I 0.1). T h e spec i f ic l a e v o r o t a t i o n of ~ - l ac tog lobu l in solu- t i ons was o n l y c o m p l e t e l y r e v e r s i b l e , w h e n the p H w a s l o w e r e d to 0.4 a f t e r 1 hour , as c o m p a r e d to the spec i f i c l a e v o r o t a t i o n of !~-lactoglobulin m e a s u r e d 30 ra in a f te r so lub i l i za t ion (da shed l ine) . W h e n ~th.e so lu t ions w e r e k e p t for a l o n g e r p e r i o d at p H 8.9 ,the r e v e r s i b i l i t y of ~he s p e c i f i c l a evo ro - t a t ion d e c r e a s e d m o r e ~and more . Af te r 72 h o u r s ~-A y i e l d e d a precipi`t~aie al.rea~dy oa l o w e r i n g the p H be low 6.4, and a f te r 2164 ,and 384 h o u r s bo th ~-A a n d ~-B y i e l d e d i t a l r e a d y b e l o w p H 7.5.

The ex ten t of the r e v e r s i b i l i t y of the spec i f ic l a e v o r o t a t i o n w a s s h o w n to be p r o p o r t i o n a l on ly to the a m o u n t of u n d e n a t u r e d ~ - l ac tog lobu l in left

BIOCHIMIE, 1971, 53, n ° 10.

Elution volume (ml)

FI6. 5. - - Gel filtration on a Sephadex G-10g column (70 em X 3.2 cm) in Tris-HC1 buffer (pH 8.2, I 0.05) of 6-1actoglobulin A and B ( - ) kept for : (a) 30 min, (b) 24, (e) 72, (d) 120 and (e) 168 hours at 20 ° in Tris-HC1 buffer (pH 8.9, I O.1). Calibration curve ( . . . . ), (1) ¢~-haemoeyanin of Helix po- maria, (2) bovine serum albumin monomer, and (3) B-lactoglobulin A or B ; ( -- . -- . -- ) chromatogram of ~-laetoglobulin A and B kept for two weeks at 20 ° in sodium acetate-acetic acid buffer (pH 5.5, I 0.1).

The s a m p l e t aken af te r 30 m i n s h o w e d on ly u n m o d i f i e d ~- lac tog lobul in , c o m p o n e n t IV. The c h r o m a t o g r a m s af te r a l o n g e r p e r i o d i n d i c a t e d the f o r m a t i o n of at least two g r o u p s of p roduc t s ,

1090 Harry Roels, Gisble Pr~aux and Rend Lontie.

c o m p o n e n t s I + II and I I I , e lu t ing fas te r t h a n c o m p o n e n t IV. C o m p o n e n t I, e lu t ing at the v o i d v o lume , i n c r e a s e d w i t h d e n a t u r a t i o n t ime, w h i l e c o m p o n e n t IV d e c r e a s e d a c c o r d i n g to f i r s t -o rde r k ine t i c s . The ra te cons t an t s a r e 10.1 X 10-~ h o u r -1 and 7.3 X 10-a hour-1 fo r ~-A and ~-B re spec t i - ve ly , and are s imi l a r to those o b t a i n e d w i t h the p o l a r i m e t r i c s tudy of the a lka l i ne d e n a t u r a t i o n of the t w o p r o t e i n s at 20 ° in Tris-HC1 buf fer (pH 8.9, I 0.1).

~ -Lac tog lobu l in A and B, k e p t for two w e e k s at 20 ° in s o d i u m ace t a t e - ace t i c ac id buf fe r (pH 5.5, 1 0.1), d id not aggrega te at all as can be seen on Fig. 5.

Sedimentation coefficients of the chromatogra- phic components. The c h r o m a t o g r a p h i c c o m p o - n e n t I, i so la ted f r o m B-A, and the c o m p o n e n t s I-IV, i so la ted f r o m :~-B, kep t for 168 h o u r s at 20 ° in Tris-HC1 buf fer (pH 8.9, I 0.1), w e r e d ia lyzed

I I 1 1 71\ ,

o~ ~/ \ \ o so

o 2 o o , t o o 6 o o a o o hours K:X~O

Fro. 6A. - - Change in thiol content ( - - ) and in specific laevorotation at 436 nm and 20 ° ( . . . . . ) of ~-lactoglobulin A (O) and B ( o ) kept at 20 ° in Tris-HCl buffer (pH 8.9, I 0.1). Thiol determinations were performed with PMB in Tris-HC1 buffer (pH 8.2, I O.1).

Fro. 6B. - - Relation between the percentage of the increase in specific laevorotat ion and of the decrease in thiol content of ~-lactoglobulin A (©) and B (o) , calculated from Fig. 6A.

a n d l y o p h i l i z e d . C o m p o n e n t I of a lka l ine dena - t u r e d ~-A s h o w e d s2o,w = 16.8 S. T h e s20.w-values of c o m p o n e n t I and II of a lka l i ne d e n a t u r e d ~-B, a f t e r f u r t h e r p u r i f i c a t i o n on S e p h a d e x G-100, a r e 13.8 a n d 3.48 S, and tha t of c o m p o n e n t I I I and IV 2.66 and 2.34 S r e s p e c t i v e l y .

Thiol determinations. ~-Lac tog lobu l in A and B so lu t ions (23 g / l ) in Tris-HC1 buf fe r (pH 8.9, I 0.1) w e r e f i l t ra ted on Mi l l i po re f i l ter HA 0.45 ~ (Bed- ford , Mass., USA), and kep t at 20 °. The th io l t i t e r

of the so lu t ions was c o m p a r e d w i t h t h e i r spec i f i c l a e v o r o t a t i o n at 436 n m (Fig. 6A). The i n c r e a s e in l a evo ro t a t i on , f as te r fo r [~-A t h a n for ~-B, w,as a c c o m p a n i e d by a f a s t e r d e c r e a s e in th io l c o n t e n t fo r i~-A than for p-B, w h i c h r e a c h e d n e a r l y the same va lue for b o t h p r o t e i n s a f te r 900 hou r s . T h e th io l conten t , h o w e v e r , d r o p p e d to ha l f i ts i n i t i a l va lue in 125 hou r s for ~-A and 278 h o u r s fo r ~-B, w h i l e the i n c r e a s e in spec i f ic l a e v o r o t a t i o n h a d a l r e a d y r e a c h e d ha l f i ts to ta l a m o u n t a f t e r 108 and 218 hou r s r e s p e c t i v e l y . W h e n the p e r c e n t a g e of the i n c r e a s e in spec i f i c l a e v o r o t a t i o n is p lo t t ed aga ins t the p e r c e n t a g e of d e c r e a s e in th io l c o n t e n t (Fig. 6B), no s ign i f i can t d i f f e r ence r e m a i n s bet- w e e n ~-A and ~-B. B o t h p r o t e i n s s h o w e d a 50 p. 100 dec rea se in t h io l con t en t for an i n c r e a s e in spec i f ic l a e v o r o t a t i o n of about 57 p. 100.

T h i o l d e t e r m i n a t i o n s w e r e also c a r r i e d out on the four c o m p o n e n t s i so la ted on S e p h a d e x G-100 f r o m ~- lac tog lobu l in A and B, kep t for 168 h o u r s at 20 ° in Tris-HC1 bu f f e r (pH 8.9, I 0.1). No th io l s cou ld be found in the c o m p o n e n t s l - I I I w i t h PMB, not even af ter 48 h o u r s r e a c t i o n , w h e r e a s c o m p o - n e n t IV stil l c o n t a i n e d n e a r l y 2 moles S H / 3 6 000.

0 4

0

0 4

~-A

f

e

- - -1

[3-B

I. ~Oo

0 3~x~ 4 b O I . , 3 0 0 4 0 0

Elutpon volume(m;)

FIG. 7. - - Gel t i l tration on a Sephadex G-100 column (70 cm × 3.3 cm) in t r i e thy lammonium acetate-acetic acid buffer (pH 4.7, 1 0.05) of 1[3-1actoglobulin A and B kept for : (a) 30 min, (b) 24, (e) 72, (d) 120, (e) 168, and (f) 240 hours at 20 ° in Tris-HC1 buffer (pH 8.9, I 0.1) ; the pH of the samples was first lowered to 5.2 with HCI 4 M and the precipitate removed by centri- fugation.

BIOCHIMIE, 1971, 53, n ° 10.

Alkal ine denaturat ion of ~-lactoglobulin A and B. 1091

The specific laevorota t ion of components I-IlI, measured at 436 nm in Tris-HC1 buffer (pH 8.9, I 0.1) equalled near ly the final value of alkaline denatured ~-lactoglobulin,---[~1 ] 0 ~ = 170 ° to 180 ° , and that of component IV was approx ima- tely the same as for freshly solubil ized protein.

Solubility experiments. The fract ion of ~-lacto- globulin A and B, still soluble at pH 5.2 after increas ing denatura t ion time, was compared by gel filtration on Sephadex G-100 in t r ie thy lamine- acetic acid buffer (pH 4.7, I 0.05) (Fig. 7). Only the component w i th the elution Volume of ~-lacto- globulin r ema ined in the superna tan t after adjusting the pH to 5.2. Components I-III were prec ip i ta ted at this pH. With increas ing denatn- rat ion t ime the amount of mater ia l soluble at pH 5.2 decreased faster for #-A than for ~-B and obeyed f irs t-order kinetics.

Gel filtration of performic acid oxidized pro- ducts. Both nat ive prote in and ~-lactoglobnlin A and B, kept for 168 hours at 20 ° in Tris-HC1 buffer (pH 8.9, I 0.1), were oxidized wi th per- formic acid. Samples of 30 mg in 2 ml Tris-HC1 buffer (pH 8.2, I 0.05) were ch romatographed on a Sephadex G-100 column in Tris-HC1 buffer (pH 8.2, I 0.05) and the eluate was measured at 220 nm (Fig. 8).

E

b e f ~ d a b cf~ d ? , , ~ . . . . ii ,

i i O-A ! ~ ~ - B

,'i 7'

" ' u J/ ,,, . . . . . : -_

'Fro '< I t i ik i it ': ',,1 ~ I t : ' ~ J t ' - ~ I ', I \ J t I

2 0 0 3 0 0 4 0 0 2 0 0 3 0 0 4 0 0 Elutior~ volume (ml)

Fro. 8. - - Lower part : gel filtration on a Sephadex G-100 column (70 cm × 3.2 cm) in Tris-HC1 buffer (pH 8.2, l 0.05) of B-lactoglobulin A and B kept for 168 hours at 20 ° in Tris-HC1 buffer (pH 8.9, I 0.1) ( . . . . . ) and of their performic acid oxidized pro- ducts ( - - ).

Upper part : gel filtration of performic acid oxidized native [~-lactoglobulin A and B ( . . ) and elu- tion maximum of the reference proteins : (a) (~-hacmo- cyanin of Helix pomatia, (b) disulfide-dimer of bovine serum albumin, (c) monomer of bovine serum albu- min, and (d) ~-lactoglobulin A or B.

The elution pat terns of p e r f o r m i c acid treated alkaline denatured ,~-A and ~-B differed comple- tely f rom that of alkaline denatured ~-lactoglo- bulin (dashed l ine in Fig. 8), but it is very similar to that of pe r fo rmic acid oxidized native ~-laeto- globulin. Component I, charac te r i s t i c for alkaline denatured ~-lactoglobulin, and componen t IV are missing. The per formic acid oxidized products showed essential ly two elution maxima wi th a smaller elution volume than f reshly solubilized ~-lactoglobulin, as can be deduced from the elu- tion max ima of the reference pro te ins indicated in the upper par t of Fig. 8.

DISCUSSION.

Accord ing to the general reac t ion scheme, pro- posed by LINDEnSTROM-LANa [341 for the dena- turat ion of proteins and their proteolysis , and taking into account the findings cf TANFOnD et al. [4, 5] and PANTALONI [6], the data of this paper confirm that the alkal ine denatura t ion of ~-lactoglobulin A and B also proceeds through at least two steps : N ~ ~-R >-D~.

N ~ " R refers to the fast ionizat ion-l inked revers ib le conformat iona l t rans i t ion of N (the stable form of native ~-lactoglobulin) into the R-conformat ion , observed at 20 ° upon increas ing the pH f rom 6.8 to 9.25. It is accompan ied by a t ransformat ion of 15 p. 100 of the ~-structure into unorde red structure.

R ~- D i refers to the slow and i r revers ible t ransformat ion of the less stable R-form into the Di-form w h e n the solutions of ~-lactoglobulin are kept above pH 7.5 saturated wi th air. This second react ion step consists of a fu r the r t ransformat ion of the ~-structure into unorde red s t ructure (the ct-helix r ema in ing constant). A concomi tan t poly- mer iza t ion of ,~-lactoglobulin is observed by gel fi l tration on Sephadex G-100 and by ul t raeentr i fu- gation, together wi th a decrease in thiol content. The aggregates, eluting faster on Sephadex G-100 than unal tered ~-lactoglobulin, are insoluble at pH 5.2.

The results agree wi th the s tatement of KAUZ- MANN [3.5] that a denatura t ion react ion, accompa- nied by a large increase in optical rotation, consists par t ly in an extensive b reakdown of the conformat ion of the prote in molecules.

The format ion of aggregates in ~-lactoglobulin solutions, kept at room tempera tu re at about pH 8.6, has also been observed by po lyacry lamide [3'6] and starch-gel [37] e lect rophoresis , and by u l t racent r i fugat ion [21] of solutions of B-A, kept at pH 9.1 and 3 ° .

BIOCHIMIE, 1971, 53, n ° 10.

1 0 9 2 H a r r y R o e l s , Gisdle P r d a u x a n d R e n d L o n t i e .

T h e d e c r e a s e in t h i o l c o n t e n t d u r i n g t he a lka - l i n e d e n a t u r a t i o n of ~ - l a c t o g l o b u l i n A a n d B is m a i n l y due to t h e o x i d a t i o n of t h e c y s t e i n e s ide - c h a i n s b y d i s s o l v e d a i r . S p e c i a l a t t e n t i o n to t h i s h a s a l so b e e n d r a w n fo r t h e u r e a [9, 38] a n d h e a t d e n a t u r a t i o n [19~ of ~ - l a c t o g l o b u l i n . T h e t i m e d e p e n d e n t p o l y m e r i z a t i o n of ~ - l a c t o g l o b u l i n A a n d B a t a p H a b o v e 7.5 i s b r o u g h t a b o u t b y a t h i o l - d i s u l f i d e e x c h a n g e c o u p l e d to a t h i o l o x i d a - t i o n a n d is c o m p l e t e l y i n h i b i t e d b y b l o c k i n g t h e t h i o l g r o u p s [20] ; s i m i l a r r e s u l t s w e r e o b t a i n e d b y JENNESS [39] f o r t h e t h e r m o d e n a t u r a t i o n of ~ - l a c t o g l o b u l i n p lu s u - l a c t a l b u m i n .

T h e f i r s t - o r d e r k i n e t i c s of t h e i r r e v e r s i b l e c o n - f o r m a t i o n a l c h a n g e a n d of t h e d e c r e a s e in t h e c h r o m a t o g r a p h i c c o m p o n e n t c o r r e s p o n d i n g to u n a l t e r e d I~- lactoglobul in , s h o w t h a t ~-A at p H 8.9 a n d 20 ° is a b o u t t w i c e as s u s c e p t i b l e to a l k a l i n e d e n a t u r a t i o n as ~-B ; t h e d e c r e a s e in t h i o l c o n t e n t is a lso a b o u t t w i c e as fas t f o r ~[~-A as f o r ,6-B. F i r s t - o r d e r k i n e t i c s f o r t he i n c r e a s e in l a e v o r o t a t i o n h~ave b e e n r e p o r t e d b y GROVES et al. [7] for t h e a l k a l i n e d e n a t u r a t i o n of p o o l e d ~ - l a c t o g l o b u l i n a n d b y McKENZlE a n d SAWYER [21], w h o f o u n d t h a t t h e r a t e of i n c r e a s e in l a e v o r o t a t i o n at p H 9.1 a n d 3 ° is in t h e o r d e r ~-A > ~-B > fl-C. By h e a t t r e a t m e n t a t 70 ° a n d p H 6.7, on t h e c o n t r a r y , ~-B w a s f o u n d to be m o r e s u s c e p t i b l e t h a n fl-A [18] a n d 6-C e v e n m o r e t h a n ~-B[19] .

W e do no t h a v e a n y e v i d e n c e t h a t t he p o l y m e - r i z a t i o n r e a c t i o n o c c u r s v ia t h e s u b n n i t s of ~-lac- t o g l o b u l i n , b u t t a k e n i n t o a c c o u n t t h e e n h a n c e d d i s s o c i a t i o n of ~ - l a c t o g l o b u l i n i n a l k a l i n e m e d i u m [2, 3] , t h e t i m e d e p e n d e n t f o r m a t i o n of a g g r e g a t e s c o u l d p r o c e e d v ia t h e m o r e d e n a t u r a b l e s u b u n i t in t h e R - c o n f o r m a t i o n . T h i s r e a c t i o n m e c h a n i s m r e q u i r e s f o r ~-A, t h a t i s m o r e s u s c e p t i b l e to a l k a l i n e d e n a t u r a t i o n t h a n ~-B, a h i g h e r d e g r e e of d i s s o c i a t i o n i n to s u b u n i t s t h a n ~-B. T h i s w a s i n d e e d f o u n d b y gel f i l t r a t i o n on S e p h a d e x G-100 a t a p H a b o v e 7.0 [17] a n d b y s e d i m e n t a t i o n equ i - l i b r i u m s t u d i e s at p H 7.5 a n d 20 ° [21].

Aeknawledgements.

We wish to t h a n k the I n s t i t u u t to t Aanmoediging van het Wetenschappe l i jk Onderzoek in Ni jverheid en Landbouw and the Comit6 voor Wetenschappe l i jk en Technisch Zuivelonderzoek for research grants .

We are also grateful to Mrs. J. HEREMANS for the careful p repara t ion of [3-1actoglobulin A and B and to Mr. G. VAN AERSCHOT for his ski lful assis tance in the u l t racent r i fuge exper iments .

Rr~SUM~.

La t r ans i t ion conformat ionne l l e N ~ " R des ~-lactoglobulines, qui se produi t de mani6re ins t an - tan(!e et rdversible h 20 ° entre pH 6,8 et 9,25, se carac- tdrise par un changemen t de 15 p. 100 de la s t ruc ture en s t ruc ture d~sordonn6e, le pourcentage de l'h61ice o. r e s t an t constant .

Au-dessus de pH 7,4 et eu prdsence d 'a i r la Idvorota- t ion des solut ions de .B-A et de ~-B croft en fonct ion du temps et ce d ' a u t a n t plus r ap idement que le pH est ~lev6. Cette ddna tu ra t ion alcal ine provoqne une t r ans fo rma t ion irrdversible de la s t ructure ~ en struc- ture d6sordonnde, t and i s que le pourcentage d'h61ice n ne var ie gu6re.

A pH 8,9 et 20 ° le changement confo rmat ionne l irr6ve:rsible s 'accompagne de la fo rmat ion d 'au moths deux groupes de composants s 'd luant plus r ap idemen t sur Sephadex G-100 que la [J-lactoglobuline comme telle. Ces composants sont insolubles h pH 5,2. Une d iminu t ion prononcde du contenu en groupes th iol est dgalement observde.

L 'accroissement de ldvorotat ion, la po lymdr isa t ion et la d iminu t ion du contenu en groupes thiol, dus h la d5na tura t ion alcaline, se produisent h pH 8,9 et 20 ° envi ron deux fois plus rap idement pour la [3-A que pour la ,~-B.

ZUSAMMENFASSUNG.

Die ~-Lactoglobuline A und B unter l iegen bei 20 ° im pH-Bereich von 6,8 bis 9,25 einem u n m i t t e l b a r e n und reversiblen N ~ ~ R -Konformat ions i ibergang. W~ihrend bet diesem Vorgang der Gehalt an a-Helix kons t an t bleibt, beobach te t man bet den Bereichen mi t ~ -S t ruk tu r eine Uber f i ih rung in ungeordnete Konfor- ma t ion im Ausma/~ von 15 p. 100.

Bet luftges/ i t t igten LSsungen der beiden Pro te ine steigt oberha lb pH 7,4 die Laevorota t ion mi t der ZeiL und zwar um so schneller , je hSher der pH-~Tert ist. Diese alkal ische Dena tu r i e rung stel l t eine irreversible Umwandlung der [~-Struktur in ungeordnete Konfor- mut ton dar, der Gehal t an u-Helix wird auch hierbei kaum veriindert.

Die i r revers ible Konformat ions / inderung bet 20 ° und pH 8,9 ist mi t der En t s t ehung von mindes tens zwei Komponentengruppen verbunden, deren Elu t ionsvolu- mina an Sephadex G-100 kle iner sind als die der unverSnder ten ~-Lactoglobuline. Der Thio l -Gehal t der Dena tu r i e rungsproduk te ist gegeniiber dem der Aus- gangsmater ia l i en s ignif ikant erniedrigt , die Subs tan- zen sind bet pH 5,2 unlSsl ieh.

Beim [J-Lactoglobulin A ver laufen die durch alka- l ische Dena tur ie rung bet 20 ° und pH 8,9 bewirk te Z u n a h m e der Laevorota t ion , die Abnahme des Sulfhy- dry lgruppen-Gehal tes und die Po lymer i sa t ion fas t doppel t so schnell wie beim ~-Lactoglobulin B.

REFERENCES.

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2. GEORGES, C. and GUINAND, S . - - J. Chim. phys., 1960, 57, 606.

BIOCHIMIE, 1971, 53, n ° 10.

Alkaline denaturation of f~-lactoglobulin A and B. 1093

3. GEORGES, C., GUINAND, S. and TONNELAT, J. - - Bio- ch im. b i o p h y s . Acta, 1962, 59, 737.

4. TANFORD, C., BUNVILLE, L. and NOZAK1, Y. - - J. A m . chem. Sac., 1959, 81, 4032.

5. TANFORD, C. and TAGGART, V . - - J. A m . c h e m . Sac., 1961, 83, 1634.

6. PANTALONI, D . - - C . r . hebd. Sdanc. Acad. Sci., Par is , 1961, 252, 2459.

7. GRovEs, M., HIPP, N. and McMEEKIN, T. - - J. A m . chem. Sac., 1951, 73, 2790.

8. SCHELLMAN, J. - - C. r. Tray. Lab. Car lsberg , Sdr. chim. , 1958, 30, 395.

9. KORSGAARD CI-IRISTENSEN, L. - - C. r. Trao. Lab. Carlsberg, Sdr. ch im. , 1952, 28, 37.

10. DUPONT, M. - - B i o c h i m . b iophgs . Ac ta , 1965, 102, 500.

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