- Email: [email protected]
Action of N-bromosuccinimide on sperm whale myoglobin
BIOCHI~{ICA E T BtOPHYSICA ACTA
309
BBA 25o25
A C T I O N O F N - B R O M O S U C C I N t M I D E ON S P E R M W H A L E M Y O G L O B I N G. ]. S. RAO AND H. R. CAMA
Department of Biochemistry, Iudia~ Institute of Scie~ce, Ba~ga~ore (I~dia) (Received A u g u s t i9th, I963)
SUMMARY
Previously, it was reported from this laboratory that the heine groups of hemoglobin are "buried" within globin at pH 4.o and not dissociated, on the basis of the obligatory requirement of urea for the reaction of N-bromosuccinimide with the heine groups of hemoglobin at pH 4.o, and also on the basis of the "normalization" of the spectrum of hemoglobin at this p H in the presence of urea or sucrose. In the present study, it has been shown that the behaviour of sperm whale myoglobin with respect to its reaction with N-bromosuecinimide arid with respect to spectral "normalization" in urea or sucrose are essentially similar to that of hemoglobin. It has also been demonstrated that the spectral "normalization" obtained with crystalline heroin is not identical with that obtained with either hemoglobin or myoglobin. The bearing of the results of the present study on the earlier work on hemoglobin is indicated.
INTRODUCTION
In a previous paper, we reported 1 a spectrophotometric study of the reaction of the heme groups of hemoglobin with NBS at pH 4.o and suggested that the Ileme groups m a y not be dissociated from globin at this pH. Further, the spectral "normalization'" appeared to be restricted to a narrow range of p H around 4.o. In view of the similarity between the folded structures of hemoglobin and myoglobin in the solid state ~ and in aqueous solutions a and also in the similarity of behaviour on acid denaturation< 5 the present study was undertaken to examine if the behaviour of myoglobin towards NBS was similar to that of hemoglobin. MATERIALS AND METHODS
Crystalline sperm whale metmyoglobin was kindly given by Dr. A. B. Edrnundson, University of Cambridge, England. It was similar in purity to the materia! employed by the Cambridge group for structural studies. Crystalline heroin (Hoffmann LaRoche) was dissolved in methanol Anaivtic Reagent)-acetic acid (96:4, v % to give a 3" IO-~ M solution. Abbreviation: NBS, N-bromosuccinimide. * P r e s e n t address: Dr. H. R. Cama, P h . D . , D. Sc. (Liverpool), F.R.I.C. chemistry, I n d i a n I n s t i t u t e of Science. BangaIore-Iz (India
Departmen-~ of Bio-
Biochim. Bio4)hvs. Acta. 80 (I964) 3o9-3~6
310
G. S. J. RAO, H. ~R. CAMA
Concentration of metmyoglobin was determined spectrophotometrically using ~ ~ 16-lO 4 at 408 mff and at p H 5.6 (ref. 5). All other materials and methods were similar to those employed earlier 1. RES ULTS
Estimation of tryptophan: The results of the "titration" of tryptophan residues of myoglobin at p H 4.0 and in 8 M urea are recorded in Fig. I. As with hemoglobin 1, a lag is observed before the t r y p t o p h a n residues are attacked, and thereafter, tile decrease in absorbancy is linear with the addition of NBS. The maximal decrease in 35
4
0
1
3O
o* E o 00 oJ
*6 ro
>~
i
o ×
25
L0
2 ~£a E o
20
I I 3.2 (5.4 moles NBS/mole
I 9.6
12.8
Mb
Fig. I. " T i t r a t i o n " of t r y p t o p h a n g r o u p s of m y o g l o b i n a t pFI 4.0 in 8 M u r e a - o . i M s o d i u m acetate medium.
absorbancy corresponds to about 2. 4 tryptophan residues per mole of myogiobin, a value close to that obtained b y EDMUNDSOS A~D HIRSs. About 2.5 moles of NBS are required to "titrate" one tryptophan residue, similar to the value observed with hemoglobin 1.
Changes is the absorption spectrum of myoglobin after the addition of NBS at pH 4.o: When NBS was added to myoglobin in excess to that required for the "titration" of t r y p t o p h a n residues, the accompanying spectral changes were similar to those observed with hemoglobin 1 (Fig. 2 ; a, b and c). The spectrum of myoglobin at 2 moles of NBS did not show any significant change at all wavelengths (Fig. 2a). At approximately 8 moles of NBS, the spectra in the ultraviolet region were characteristic of oxindole groups1,7, s. In addition, at 8-mole level of NBS, there was a definite decrease in the intensity of the Soret extinction (Fig. 2b) but not in the 5oo-7oo-mff region (Fig. 2c.). At 4o moles of NBS all the absorption maxima in the visible region disappeared. Urea was obligatory for the reaction. Biochim. Biophys. Acla, 86 (1964) 3 o 9 - 3 1 6
ACTION OF N-BROMOSUCCINIMIDE ON MYOGLOBIN
]lll
"Titration" of heine moiety at pH 4.o and at pH 7.0 : The results o5 t h e " t i t r a t i o n ' of t h e heme m o i e t y a g a i n s t N B S are s u m m a r i z e d in Fig. 3. A t p H 4.o a n d in 8 M u r e a tim a b s o r b a n c y did n o t show a n y change untii a b o u t 32 F
(Cl)
16 f
(b)
24
8
4
, 240
280 rn#
2
~
[["-"'-U×-×-×-.~-, ( --,-,~--~-~-x-×-~-x--I ,->~_x. . . . . . . .
320 360
400 m/a
440
480 500
540
580 rnp
620
660
1
700
~'ig. 2. Spectral changes in m e t m y o g l o b i n after the addition of NBS. (l~igures r e d r a w n from recordings of a B e c k m a n D B spectrophotometer.) @ @, No NBS; @ @, 2 moles N B S / m o l e myoglobin; O O, 8 moles N B S / m o l e m y o g l o b i n ; × × , 4 ° moies N B S / m o l e myoglobin.
12
~t
pH
0[ o-~a
L_
t ~6
I
24 rnoies NBS/mole Mb
7.0
{
a2
4'0
Fig. 3. Reaction of the heine group of m y o g l o b i n w i t h NBS. The reaction was followed at 400 m # at p H 4.0 and at 4o8 mff at p H 7.o.
8 moles of N B S were a d d e d a n d t h e r e a f t e r t h e decrease in a b s o r b a n c y at 400 mb~ was v e r y striking. The presence of u r e a was o b ! i g a t o r y at this p H as the solutions b e c a m e t u r b i d when it was omKted. S t r i c t l y for reasons of comparison with h e m o g l o b i n 1. the readings were recorded after 4 h of reaction a t 24-26 °. About 14 moles of N B S were Biochim. Biophys. Acta, 86 (I964) 3o9-316
312
G. S. J . R A O , H . R. CAMA
needed for 5o % reduction in heine absorption. Allowing for the 6 moles of NBS (approx.) needed before any change in absorbancy at 400 m/~ was noted, about 8 moles of NBS were, therefore, required for a 50 % reduction in heine absorption. TABLE EFFECT OF
I
NBS ON THE RELEASE OF INORGANIC IRON FROM MYOGLOBIN
O.I # m o l e o f m y o g l o b i n i n i . o m l o f 8 M u r e a - o . i M s o d i u m a c e t a t e b u f f e r ( p H 4.0) w a s t r e a t e d w i t h d e s i r e d v o l u m e s of o . i iV[ N B S a t 2 4 - 2 6 °. A f t e r 4 h, t h e v o l u m e w a s m a d e t o 2 . 0 m l a n d 2.0 m l of I o % (w/v) t r i c h l o r o a c e t i c a c i d w e r e a d d e d . T h e c o n t e n t s w e r e c e n t r i f u g e d a n d i . o - m l a l i q u o t of t h e s u p e r n a t a n t a n a l y s e d f o r i r o n a s i n r e f . i.
Moles NBS/mole of myoglobin
% Inorganic iron released
o
o
2
o
8
27
16
57
24 32
95 IOO
The reaction at p H 7.0 was followed at 4o8 m/z. At this p H also, NBS could react with the heme group, but urea was not obligatory. The lag of 6 moles observed at p H 4.0 was not encountered at p H 7.0. There is an obvious similarity in the course of i no urea
16-
,8 rio u r e a
16
12
12 pH 4 . 0 0.1M acetate
£,;
-70 % sucrose
8
0
4 0.1M acetate
\ 0 360
I 380
I 400 mp
I 420
I 440
F i g . 4. " N o r m a l i z a t i o n " of m ' y o g l o b i n s p e c t r u m a t p H 4 . 0 i n 8 M u r e a . F o r d e t a i l s , see t e x t .
0 .360
I 380
l 400 m~
I 420
I 440
F i g . 5. E f f e c t of s u c r o s e o n t h e s p e c t r u m m y o g l o b i n a t p i l l 4.0.
o£
Biochim. Biophys. Acted, 8 6 (1964) 3 o 9 - 3 1 6
ACTION OF I~r-BROMOSUCCINiMIDE ON MYOGLOBIN
,~I3
reaction of NBS with hemoglobin (ref. I, Fig. 5) and myoglobin at both the pH values. At pH 7.o, about 22 moles are needed for a 50 % reduction in heine absorption, Release of inorgct~ic iron after the addition of N B S : Addition of NBS t o myogtobin at pH 4-o caused the release of inorganic iron from the heine group (Table I). Almost all the iron is released at a level o~ 25 moles of NBS when, correspondingly, the heine absorbancy also reaches a near minimum (Fig. 3). Effect of urea on the release of inorganic iron : The effect of urea on the release of inorganic iron was studied only in 4 M and 8 M urea media at a constant NBS level of 32 moles per mole of myoglobin. Though about 7 ° % of iron was released in 4 M urea almost the entire amount of iron was released in 3 M urea, suggesting a relationship between urea concentration and the release of iron. A bsorptio~ spectrum of myogZobin in 8 M urea: The spectrum of myoglobin in 8 ?~{ urea at p H 4.o was distinctly different from the spectrum in the absence of urea (Fig. 4). For comparison, the spectrum of the same sample of myoglobin at pH 5,6 and at p H 6.8 were also recorded. It is at once apparent that the absorbancy of myoglobin at pH 4.o in 8 M urea (at 4oo m~) is equal to that at pH 6.8 and very nearly equal to that at p H 5.6 (at 4o8 m~). One notable point is that the absorption maximum is shifted to 4oo m/, at p H 4.o in 8 M urea from 408 m/~ as in hemoglobint On the other hand, the spectrum at p H 4.o in the absence of urea shows an ill-defined maximum at 4o8 m/,, but the absorbancy is only about one fourth of that in the presence of urea. Abso@tio¢~ spectrum of myoglobin in s,rzcrose ~edium : The spectrum of myoglobin at pH 4.o in 7 ° % sucrose medium had a maximum at 4o8m~ with
16
12
ij
50 % me'~hanol
14
\
0 0 I o
"7 o
12
%
\
% ko
8
10-
\
8 2.0
I
3.0
I
pN
4.0
! ~o 5,0
0
I
Fig. 6. Sorer absorbancy of metmyoglobin in 8 iK urea as a f u n c t i o n of p H .
Acetate buffer I I I 380 400 420 m#
.~
440
Fig. 7" A b s o r p t i o n s p e c t r u m of heroin a t p H 4~o in different m e d i a .
Biochim. Biophys. Acla, 86 (i964) 3o9-316
314
c.s.j.
RAO, H. R. CAMA
Heine absorption as function of pH in 8 M urea: The relationship between the Soret absorbancy of myoglobin in 8 M urea and p H 4.0 is shown in Fig. 6. I t is obvious that the pattern of "normalization" of myoglobin spectrum is similar to that of hemoglobin (see ref. I, Fig. IO). As seen with hemoglobin 1, the heine absorption in myoglobin is "normalized" around p H 4.0 to reach complete intensity at 400 m/~ (aM = 15.5"1o4). Whereas a steady increase in heine absorbaney was observed between p H 2.5 and 3.0 in the case of hemoglobin, there is a decreasing trend in this p H range with myoglobin. This perhaps is due to the differences between the protein moieties of the two pigments. Absorption spectrun¢ of heroin at pH 4.o: I t is well established that heroin is aggregated in aqueous solutions% ~°. Heroin is not aggregated in freshly prepared dilute solutions, but does so on standing 11. Several agents like ethanoP 2, ethylene glycol 1~ and caffeine 14, break the aggregates of heroin as evidenced by the appearence of the well defined absorption maxima, particularly in the Soret region. I t is possible that the absence of a sharp Soret m a x i m u m at p H 4.0 is due to the aggregated nature of the heroin dissociated from globin at this p H and that regeneration of the spectrum is caused b y the breaking of aggregates in the presence of urea or sucrose. To test this possibility, spectra of heroin were recorded in different media at p H 4.0 (Fig. 7). The spectrum in o.I M acetate buffer-5o % methanol shows a sharp m a x i m u m at 400 m/~ with e• = 16- IO~, a value obtained b y SMITH1~, MAEHLY AND AKESSON12, suggesting a complete disaggregation of hemin in this medium. But the eM in 8 M urea is about half this value and in o.I M acetate and 7 ° % sucrose-o.I M acetate, the spectra do not show any m a x i m u m ill the Soret region. Thus, the spectral "normalizations" obtained in 8 M urea or 7 ° % sucrose with hemoglobin and myoglobin are not identical to those obtained with heroin. DISCUSSION
When the criteria employed to study the nature of heme-globin linkage in hemoglobin 1 were applied to my0globin, essentially similar results were obtained. As in hemoglobin, the NBS-myoglobin reaction can be divided, though not clearly, into three stages : a, reaction with about 2 moles of NBS, not resulting in a change in indole absorbancy (Fig. i) ; b, the NBS-tryptophan reaction requiring about 6 moles of NBS (Fig. I) ; c, tile NBS-heme reaction causing a total change in the spectrum of the heme group but only in the presence of urea (Figs. 2b and 2c). The lag in the case of hemoglobin was interpreted to be due to the reaction of NBS with the - S H groups 1. But as myoglobin does not contain - S H groups, the nature of this reaction in Stage a, is unknown at present. The reactions at Stages b and c could not be separated clearly, as shown b y the large decrease in the Soret extinction, though the shape of the spectrum is not drastically altered (Fig. 2b). The NBS-heme reaction (Stage c) as indicated by the total alteration in the spectrum suggests profound changes in the structure of the heme groups. Urea was obligatory for the "normalization" of the spectrum as it was for tile release of inorganic iron (Table II). Hence, it can be concluded that in myoglobin also, heme is "buried" and is available for reaction with NBS only when "denatured" with urea. The heme group could react with NBS at p H 7.o in the absence of urea as it is situated on the surface of the molecule at this p H 2, but urea was obligatory for the reaction at p H 4.o. Biochim. Biophys. Acta, 86 (1964) 3o9-316
ACTION
OF .TV-BROMOSUCCINIMIDE
ON MYOGLOBIN
315
About 8 mo]es of NBS are needed for 50 % reduction in heine absorbancy per heine group at p H 4.o, similar to 7 d moles needed in hemogIobin. At pH 7.o, about z2 moles of NBS are needed for 50 To reaction in myoglobin (Fig. 3), whereas about 4o moles are needed in hemoglobin (per heine group). In the earlier study 1 carbonmonoxyhemoglobin was employed, which has heme in the ferrous state and iron bound to carbonmonoxide in a covalent linkage, whereas in metmyogiobin, the heine group is in the ferric form with iron in the ionic linkage. These differences may be responsible for the variation in the amount of NBS consumed per heine group at p H 7.0. The amount of NBS consumed for half reaction is the same for both the proteins at pH 4.0 in the presence of urea as the heine groups in both the proteins are in the ferric state and any interference by the protein moiety would perhaps be reduced in the presence of urea. Myoglobin undergoes a conformational change in acidic media as shown by the large decrease in helical content ~ but without any decrease in molecular weight as it has only one peptide chain. Thus, it may be that such a conformationat change is responsible for the low intensity of the spectrum in the Sorer region at p H 4.0 and also for the failure of NBS to react in the absence of urea with the heme group. But in the presence of urea at p H 4.o the spectrum is almost completely "normalized" (s~ = I5.5" Io 4 at 4oo mt~) and also the heine group becomes completely reactive towards NBS (Fig. ~, Table I). These observation are essentially similar to our earlier observations on hemoglobin 1 and hence it may be concluded that i~ myogtobin also heme is "buried" within globin at p H 4.o. Further, the pH region of "normalization" of spectrum in 8 M urea is also similar to that of hemoglobin. While a steady increase in absorbancy was observed between p H ~.5 and 3.o with hemoglobin, an initial decrease is observed with myoglobin (Fig. 6). The reasons for this difference are not clear at present, but it is probably governed by the differences between the protein moieties. The evidence that crystalline heroin does not undergo spectral changes simiiar to hemoglobin or myoglobin in urea or sucrose shows that spectral changes are due to the proteins and not due to the dissociated prosthetic group (Fig. 7). Moreover, the extent of "normalization" in both the proteins in 7o To sucrose (@ Fig. 7 with Fig. 9 of ref. I) and the narrow pH range of "normalization" in contrast to the appearance of sharp Soret maximum of hemin even in H2SO4oethanol mixtures~% suggest that the phenomenon may not be one of mere disaggregation of hemin. Since myoglobin shows a behaviour essentially similar to hemoglobin% it is pertinent to suggest, on the basis of analogy, that in hemoglobin the interaction between the peptide chains may not be necessary for spectral "normalization". That is, it is probable that the spectral changes are manifested by each peptide chain independent of the other and it is the quarter molecule of hemoglobin which exhibits the above properties. It can be recalled that at pH 4.3 in the absence of urea, the electron micrographs show that the shape of the subunit of hemoglobin is similar to that of myoglobinl% ACKNOWLEDGEMENT This investigation was supported by a grant from the Council of Scientific and Industrial Research, New Delhi, India. f3iochim. Biophys. Ads, 86 (~964) 309 316
316
G.s.j.
RAO, H. R. CAMA REFERENCES
1 G. J. S. RAO AND H . R . CAMA, Biochim. Biophys. Acta, 71 (1963) 139. 2 M. F. PERUTZ, Protein Struture and Function, Brookhaven Syrup. on Biology, Vol. 13,196o, p. 165. :~ S. BEYCHOCK AND E . 1~_. BLOUT, J. Mol. Biol., 3 (1961) 769 • a S. BEYCHOCK, C. D E L o z E AND E . :R. BLOUT, J. Mol. Biol., 4 (I962) 421. 5 E . BRESLOW AND F. R . N. GURD, J. Biol. Chem., 237 (1962) 3716 A. B. EDMUNDSON AND C. H . W . HIRS, Nature, 19o (196I) 704 . 7 T. PETERS, Compt. Rend. Tray. Lab. Carlsberg, 29 (1959) 2278 L. K. ~.AMACHANDRAN AND B. WITKOP, J. Am. Chem. Soc., 81 (1959) 4 °28. " J. SHACK AND W . M. CLARK, J. Biol. Chem., 171 (1947) 143. 10 ]~_, I. WALTER, J. Biol. Chem., 196 (1952) 151. 11 y . INADA AND K. SHIBATA, Biochem. Biophys. Res. Commun., 9 (1962) 323 . l i A. C. MAEHLY AND A. AKESSON, Aeta Chem. Scand., 12 (1958) 1259. 13 M. H . SMITH, Biochem. J., 73 (1953) 90. la j . KEILIN, Biochem. dr., 37 (1943) 281. 15 O. LEVlN, J. Mol. Biol., 6 (1963) 158.
Biochim. Biophys. Acta, 86 (1964) 3 o 9 - 3 1 6
309
BBA 25o25
A C T I O N O F N - B R O M O S U C C I N t M I D E ON S P E R M W H A L E M Y O G L O B I N G. ]. S. RAO AND H. R. CAMA
Department of Biochemistry, Iudia~ Institute of Scie~ce, Ba~ga~ore (I~dia) (Received A u g u s t i9th, I963)
SUMMARY
Previously, it was reported from this laboratory that the heine groups of hemoglobin are "buried" within globin at pH 4.o and not dissociated, on the basis of the obligatory requirement of urea for the reaction of N-bromosuccinimide with the heine groups of hemoglobin at pH 4.o, and also on the basis of the "normalization" of the spectrum of hemoglobin at this p H in the presence of urea or sucrose. In the present study, it has been shown that the behaviour of sperm whale myoglobin with respect to its reaction with N-bromosuecinimide arid with respect to spectral "normalization" in urea or sucrose are essentially similar to that of hemoglobin. It has also been demonstrated that the spectral "normalization" obtained with crystalline heroin is not identical with that obtained with either hemoglobin or myoglobin. The bearing of the results of the present study on the earlier work on hemoglobin is indicated.
INTRODUCTION
In a previous paper, we reported 1 a spectrophotometric study of the reaction of the heme groups of hemoglobin with NBS at pH 4.o and suggested that the Ileme groups m a y not be dissociated from globin at this pH. Further, the spectral "normalization'" appeared to be restricted to a narrow range of p H around 4.o. In view of the similarity between the folded structures of hemoglobin and myoglobin in the solid state ~ and in aqueous solutions a and also in the similarity of behaviour on acid denaturation< 5 the present study was undertaken to examine if the behaviour of myoglobin towards NBS was similar to that of hemoglobin. MATERIALS AND METHODS
Crystalline sperm whale metmyoglobin was kindly given by Dr. A. B. Edrnundson, University of Cambridge, England. It was similar in purity to the materia! employed by the Cambridge group for structural studies. Crystalline heroin (Hoffmann LaRoche) was dissolved in methanol Anaivtic Reagent)-acetic acid (96:4, v % to give a 3" IO-~ M solution. Abbreviation: NBS, N-bromosuccinimide. * P r e s e n t address: Dr. H. R. Cama, P h . D . , D. Sc. (Liverpool), F.R.I.C. chemistry, I n d i a n I n s t i t u t e of Science. BangaIore-Iz (India
Departmen-~ of Bio-
Biochim. Bio4)hvs. Acta. 80 (I964) 3o9-3~6
310
G. S. J. RAO, H. ~R. CAMA
Concentration of metmyoglobin was determined spectrophotometrically using ~ ~ 16-lO 4 at 408 mff and at p H 5.6 (ref. 5). All other materials and methods were similar to those employed earlier 1. RES ULTS
Estimation of tryptophan: The results of the "titration" of tryptophan residues of myoglobin at p H 4.0 and in 8 M urea are recorded in Fig. I. As with hemoglobin 1, a lag is observed before the t r y p t o p h a n residues are attacked, and thereafter, tile decrease in absorbancy is linear with the addition of NBS. The maximal decrease in 35
4
0
1
3O
o* E o 00 oJ
*6 ro
>~
i
o ×
25
L0
2 ~£a E o
20
I I 3.2 (5.4 moles NBS/mole
I 9.6
12.8
Mb
Fig. I. " T i t r a t i o n " of t r y p t o p h a n g r o u p s of m y o g l o b i n a t pFI 4.0 in 8 M u r e a - o . i M s o d i u m acetate medium.
absorbancy corresponds to about 2. 4 tryptophan residues per mole of myogiobin, a value close to that obtained b y EDMUNDSOS A~D HIRSs. About 2.5 moles of NBS are required to "titrate" one tryptophan residue, similar to the value observed with hemoglobin 1.
Changes is the absorption spectrum of myoglobin after the addition of NBS at pH 4.o: When NBS was added to myoglobin in excess to that required for the "titration" of t r y p t o p h a n residues, the accompanying spectral changes were similar to those observed with hemoglobin 1 (Fig. 2 ; a, b and c). The spectrum of myoglobin at 2 moles of NBS did not show any significant change at all wavelengths (Fig. 2a). At approximately 8 moles of NBS, the spectra in the ultraviolet region were characteristic of oxindole groups1,7, s. In addition, at 8-mole level of NBS, there was a definite decrease in the intensity of the Soret extinction (Fig. 2b) but not in the 5oo-7oo-mff region (Fig. 2c.). At 4o moles of NBS all the absorption maxima in the visible region disappeared. Urea was obligatory for the reaction. Biochim. Biophys. Acla, 86 (1964) 3 o 9 - 3 1 6
ACTION OF N-BROMOSUCCINIMIDE ON MYOGLOBIN
]lll
"Titration" of heine moiety at pH 4.o and at pH 7.0 : The results o5 t h e " t i t r a t i o n ' of t h e heme m o i e t y a g a i n s t N B S are s u m m a r i z e d in Fig. 3. A t p H 4.o a n d in 8 M u r e a tim a b s o r b a n c y did n o t show a n y change untii a b o u t 32 F
(Cl)
16 f
(b)
24
8
4
, 240
280 rn#
2
~
[["-"'-U×-×-×-.~-, ( --,-,~--~-~-x-×-~-x--I ,->~_x. . . . . . . .
320 360
400 m/a
440
480 500
540
580 rnp
620
660
1
700
~'ig. 2. Spectral changes in m e t m y o g l o b i n after the addition of NBS. (l~igures r e d r a w n from recordings of a B e c k m a n D B spectrophotometer.) @ @, No NBS; @ @, 2 moles N B S / m o l e myoglobin; O O, 8 moles N B S / m o l e m y o g l o b i n ; × × , 4 ° moies N B S / m o l e myoglobin.
12
~t
pH
0[ o-~a
L_
t ~6
I
24 rnoies NBS/mole Mb
7.0
{
a2
4'0
Fig. 3. Reaction of the heine group of m y o g l o b i n w i t h NBS. The reaction was followed at 400 m # at p H 4.0 and at 4o8 mff at p H 7.o.
8 moles of N B S were a d d e d a n d t h e r e a f t e r t h e decrease in a b s o r b a n c y at 400 mb~ was v e r y striking. The presence of u r e a was o b ! i g a t o r y at this p H as the solutions b e c a m e t u r b i d when it was omKted. S t r i c t l y for reasons of comparison with h e m o g l o b i n 1. the readings were recorded after 4 h of reaction a t 24-26 °. About 14 moles of N B S were Biochim. Biophys. Acta, 86 (I964) 3o9-316
312
G. S. J . R A O , H . R. CAMA
needed for 5o % reduction in heine absorption. Allowing for the 6 moles of NBS (approx.) needed before any change in absorbancy at 400 m/~ was noted, about 8 moles of NBS were, therefore, required for a 50 % reduction in heine absorption. TABLE EFFECT OF
I
NBS ON THE RELEASE OF INORGANIC IRON FROM MYOGLOBIN
O.I # m o l e o f m y o g l o b i n i n i . o m l o f 8 M u r e a - o . i M s o d i u m a c e t a t e b u f f e r ( p H 4.0) w a s t r e a t e d w i t h d e s i r e d v o l u m e s of o . i iV[ N B S a t 2 4 - 2 6 °. A f t e r 4 h, t h e v o l u m e w a s m a d e t o 2 . 0 m l a n d 2.0 m l of I o % (w/v) t r i c h l o r o a c e t i c a c i d w e r e a d d e d . T h e c o n t e n t s w e r e c e n t r i f u g e d a n d i . o - m l a l i q u o t of t h e s u p e r n a t a n t a n a l y s e d f o r i r o n a s i n r e f . i.
Moles NBS/mole of myoglobin
% Inorganic iron released
o
o
2
o
8
27
16
57
24 32
95 IOO
The reaction at p H 7.0 was followed at 4o8 m/z. At this p H also, NBS could react with the heme group, but urea was not obligatory. The lag of 6 moles observed at p H 4.0 was not encountered at p H 7.0. There is an obvious similarity in the course of i no urea
16-
,8 rio u r e a
16
12
12 pH 4 . 0 0.1M acetate
£,;
-70 % sucrose
8
0
4 0.1M acetate
\ 0 360
I 380
I 400 mp
I 420
I 440
F i g . 4. " N o r m a l i z a t i o n " of m ' y o g l o b i n s p e c t r u m a t p H 4 . 0 i n 8 M u r e a . F o r d e t a i l s , see t e x t .
0 .360
I 380
l 400 m~
I 420
I 440
F i g . 5. E f f e c t of s u c r o s e o n t h e s p e c t r u m m y o g l o b i n a t p i l l 4.0.
o£
Biochim. Biophys. Acted, 8 6 (1964) 3 o 9 - 3 1 6
ACTION OF I~r-BROMOSUCCINiMIDE ON MYOGLOBIN
,~I3
reaction of NBS with hemoglobin (ref. I, Fig. 5) and myoglobin at both the pH values. At pH 7.o, about 22 moles are needed for a 50 % reduction in heine absorption, Release of inorgct~ic iron after the addition of N B S : Addition of NBS t o myogtobin at pH 4-o caused the release of inorganic iron from the heine group (Table I). Almost all the iron is released at a level o~ 25 moles of NBS when, correspondingly, the heine absorbancy also reaches a near minimum (Fig. 3). Effect of urea on the release of inorganic iron : The effect of urea on the release of inorganic iron was studied only in 4 M and 8 M urea media at a constant NBS level of 32 moles per mole of myoglobin. Though about 7 ° % of iron was released in 4 M urea almost the entire amount of iron was released in 3 M urea, suggesting a relationship between urea concentration and the release of iron. A bsorptio~ spectrum of myogZobin in 8 M urea: The spectrum of myoglobin in 8 ?~{ urea at p H 4.o was distinctly different from the spectrum in the absence of urea (Fig. 4). For comparison, the spectrum of the same sample of myoglobin at pH 5,6 and at p H 6.8 were also recorded. It is at once apparent that the absorbancy of myoglobin at pH 4.o in 8 M urea (at 4oo m~) is equal to that at pH 6.8 and very nearly equal to that at p H 5.6 (at 4o8 m~). One notable point is that the absorption maximum is shifted to 4oo m/, at p H 4.o in 8 M urea from 408 m/~ as in hemoglobint On the other hand, the spectrum at p H 4.o in the absence of urea shows an ill-defined maximum at 4o8 m/,, but the absorbancy is only about one fourth of that in the presence of urea. Abso@tio¢~ spectrum of myoglobin in s,rzcrose ~edium : The spectrum of myoglobin at pH 4.o in 7 ° % sucrose medium had a maximum at 4o8m~ with
16
12
ij
50 % me'~hanol
14
\
0 0 I o
"7 o
12
%
\
% ko
8
10-
\
8 2.0
I
3.0
I
pN
4.0
! ~o 5,0
0
I
Fig. 6. Sorer absorbancy of metmyoglobin in 8 iK urea as a f u n c t i o n of p H .
Acetate buffer I I I 380 400 420 m#
.~
440
Fig. 7" A b s o r p t i o n s p e c t r u m of heroin a t p H 4~o in different m e d i a .
Biochim. Biophys. Acla, 86 (i964) 3o9-316
314
c.s.j.
RAO, H. R. CAMA
Heine absorption as function of pH in 8 M urea: The relationship between the Soret absorbancy of myoglobin in 8 M urea and p H 4.0 is shown in Fig. 6. I t is obvious that the pattern of "normalization" of myoglobin spectrum is similar to that of hemoglobin (see ref. I, Fig. IO). As seen with hemoglobin 1, the heine absorption in myoglobin is "normalized" around p H 4.0 to reach complete intensity at 400 m/~ (aM = 15.5"1o4). Whereas a steady increase in heine absorbaney was observed between p H 2.5 and 3.0 in the case of hemoglobin, there is a decreasing trend in this p H range with myoglobin. This perhaps is due to the differences between the protein moieties of the two pigments. Absorption spectrun¢ of heroin at pH 4.o: I t is well established that heroin is aggregated in aqueous solutions% ~°. Heroin is not aggregated in freshly prepared dilute solutions, but does so on standing 11. Several agents like ethanoP 2, ethylene glycol 1~ and caffeine 14, break the aggregates of heroin as evidenced by the appearence of the well defined absorption maxima, particularly in the Soret region. I t is possible that the absence of a sharp Soret m a x i m u m at p H 4.0 is due to the aggregated nature of the heroin dissociated from globin at this p H and that regeneration of the spectrum is caused b y the breaking of aggregates in the presence of urea or sucrose. To test this possibility, spectra of heroin were recorded in different media at p H 4.0 (Fig. 7). The spectrum in o.I M acetate buffer-5o % methanol shows a sharp m a x i m u m at 400 m/~ with e• = 16- IO~, a value obtained b y SMITH1~, MAEHLY AND AKESSON12, suggesting a complete disaggregation of hemin in this medium. But the eM in 8 M urea is about half this value and in o.I M acetate and 7 ° % sucrose-o.I M acetate, the spectra do not show any m a x i m u m ill the Soret region. Thus, the spectral "normalizations" obtained in 8 M urea or 7 ° % sucrose with hemoglobin and myoglobin are not identical to those obtained with heroin. DISCUSSION
When the criteria employed to study the nature of heme-globin linkage in hemoglobin 1 were applied to my0globin, essentially similar results were obtained. As in hemoglobin, the NBS-myoglobin reaction can be divided, though not clearly, into three stages : a, reaction with about 2 moles of NBS, not resulting in a change in indole absorbancy (Fig. i) ; b, the NBS-tryptophan reaction requiring about 6 moles of NBS (Fig. I) ; c, tile NBS-heme reaction causing a total change in the spectrum of the heme group but only in the presence of urea (Figs. 2b and 2c). The lag in the case of hemoglobin was interpreted to be due to the reaction of NBS with the - S H groups 1. But as myoglobin does not contain - S H groups, the nature of this reaction in Stage a, is unknown at present. The reactions at Stages b and c could not be separated clearly, as shown b y the large decrease in the Soret extinction, though the shape of the spectrum is not drastically altered (Fig. 2b). The NBS-heme reaction (Stage c) as indicated by the total alteration in the spectrum suggests profound changes in the structure of the heme groups. Urea was obligatory for the "normalization" of the spectrum as it was for tile release of inorganic iron (Table II). Hence, it can be concluded that in myoglobin also, heme is "buried" and is available for reaction with NBS only when "denatured" with urea. The heme group could react with NBS at p H 7.o in the absence of urea as it is situated on the surface of the molecule at this p H 2, but urea was obligatory for the reaction at p H 4.o. Biochim. Biophys. Acta, 86 (1964) 3o9-316
ACTION
OF .TV-BROMOSUCCINIMIDE
ON MYOGLOBIN
315
About 8 mo]es of NBS are needed for 50 % reduction in heine absorbancy per heine group at p H 4.o, similar to 7 d moles needed in hemogIobin. At pH 7.o, about z2 moles of NBS are needed for 50 To reaction in myoglobin (Fig. 3), whereas about 4o moles are needed in hemoglobin (per heine group). In the earlier study 1 carbonmonoxyhemoglobin was employed, which has heme in the ferrous state and iron bound to carbonmonoxide in a covalent linkage, whereas in metmyogiobin, the heine group is in the ferric form with iron in the ionic linkage. These differences may be responsible for the variation in the amount of NBS consumed per heine group at p H 7.0. The amount of NBS consumed for half reaction is the same for both the proteins at pH 4.0 in the presence of urea as the heine groups in both the proteins are in the ferric state and any interference by the protein moiety would perhaps be reduced in the presence of urea. Myoglobin undergoes a conformational change in acidic media as shown by the large decrease in helical content ~ but without any decrease in molecular weight as it has only one peptide chain. Thus, it may be that such a conformationat change is responsible for the low intensity of the spectrum in the Sorer region at p H 4.0 and also for the failure of NBS to react in the absence of urea with the heme group. But in the presence of urea at p H 4.o the spectrum is almost completely "normalized" (s~ = I5.5" Io 4 at 4oo mt~) and also the heine group becomes completely reactive towards NBS (Fig. ~, Table I). These observation are essentially similar to our earlier observations on hemoglobin 1 and hence it may be concluded that i~ myogtobin also heme is "buried" within globin at p H 4.o. Further, the pH region of "normalization" of spectrum in 8 M urea is also similar to that of hemoglobin. While a steady increase in absorbancy was observed between p H ~.5 and 3.o with hemoglobin, an initial decrease is observed with myoglobin (Fig. 6). The reasons for this difference are not clear at present, but it is probably governed by the differences between the protein moieties. The evidence that crystalline heroin does not undergo spectral changes simiiar to hemoglobin or myoglobin in urea or sucrose shows that spectral changes are due to the proteins and not due to the dissociated prosthetic group (Fig. 7). Moreover, the extent of "normalization" in both the proteins in 7o To sucrose (@ Fig. 7 with Fig. 9 of ref. I) and the narrow pH range of "normalization" in contrast to the appearance of sharp Soret maximum of hemin even in H2SO4oethanol mixtures~% suggest that the phenomenon may not be one of mere disaggregation of hemin. Since myoglobin shows a behaviour essentially similar to hemoglobin% it is pertinent to suggest, on the basis of analogy, that in hemoglobin the interaction between the peptide chains may not be necessary for spectral "normalization". That is, it is probable that the spectral changes are manifested by each peptide chain independent of the other and it is the quarter molecule of hemoglobin which exhibits the above properties. It can be recalled that at pH 4.3 in the absence of urea, the electron micrographs show that the shape of the subunit of hemoglobin is similar to that of myoglobinl% ACKNOWLEDGEMENT This investigation was supported by a grant from the Council of Scientific and Industrial Research, New Delhi, India. f3iochim. Biophys. Ads, 86 (~964) 309 316
316
G.s.j.
RAO, H. R. CAMA REFERENCES
1 G. J. S. RAO AND H . R . CAMA, Biochim. Biophys. Acta, 71 (1963) 139. 2 M. F. PERUTZ, Protein Struture and Function, Brookhaven Syrup. on Biology, Vol. 13,196o, p. 165. :~ S. BEYCHOCK AND E . 1~_. BLOUT, J. Mol. Biol., 3 (1961) 769 • a S. BEYCHOCK, C. D E L o z E AND E . :R. BLOUT, J. Mol. Biol., 4 (I962) 421. 5 E . BRESLOW AND F. R . N. GURD, J. Biol. Chem., 237 (1962) 3716 A. B. EDMUNDSON AND C. H . W . HIRS, Nature, 19o (196I) 704 . 7 T. PETERS, Compt. Rend. Tray. Lab. Carlsberg, 29 (1959) 2278 L. K. ~.AMACHANDRAN AND B. WITKOP, J. Am. Chem. Soc., 81 (1959) 4 °28. " J. SHACK AND W . M. CLARK, J. Biol. Chem., 171 (1947) 143. 10 ]~_, I. WALTER, J. Biol. Chem., 196 (1952) 151. 11 y . INADA AND K. SHIBATA, Biochem. Biophys. Res. Commun., 9 (1962) 323 . l i A. C. MAEHLY AND A. AKESSON, Aeta Chem. Scand., 12 (1958) 1259. 13 M. H . SMITH, Biochem. J., 73 (1953) 90. la j . KEILIN, Biochem. dr., 37 (1943) 281. 15 O. LEVlN, J. Mol. Biol., 6 (1963) 158.
Biochim. Biophys. Acta, 86 (1964) 3 o 9 - 3 1 6