Properties of hemoglobin M. Unequivalent nature of the α and β subunits in the hemoglobin molecule

262

BIOCHIMICA g'l BI()PH1SICA ACTA

BBA 35266 PROPERTIES

O F H E M O G L O B I N M. U N E Q U I V A L E N T

N A T U R E O F T H E (x

A N D fl S U B U N I T S IN T H E H E M O G L O B I N M O L E C U L E *

AKIRA HAYASHI, TOM()KAZU SUZUKI, AKIRA SHIMIZU AND Y[ ICHI YAMAMUI¢.\ Third Department ()f Internal Medicine, Osaka University, ()sak~ (.Japan)

(Received May 9th, 19(~8)

SUMMARY The s t a b i l i t y of H b Mgosto n (a258Tyrf12), M Iwate (a2sTTyr/~2), MSaskatoon (6t2f126aTyr) a n d Mnyde Park (a2 f1292Tyr), which have the same kind of a m i n o acid s u b s t i t u t i o n , histidine w i t h tyrosine, a n d are s t r u c t u r a l l y homologous in r e l a t i o n to heine irons of b o t h a a n d fl s u b u n i t s of the H b molecule, were c o m p a r a t i v e l y studied. I n the reduction of the ferric heine irons of t h e a b n o r m a l s u b u n i t s b y dithionite, H b MBoston a n d M Iwate were slower in r a t e t h a n H b Msaskatoon a n d MHyae Park. In the o x i d a t i o n of the ferrous heme irons of the n o r m a l subunits b y molecular oxygen a n d b y ferricyanide, the former two hemoglobins M were faster in rate t h a n the l a t t e r two. The physicochemical properties of hemoglobins M~°-12, ~618 a n d t h e present findings indicate t h a t t h e a s u b u n i t s of t h e H b M molecule combine with ligands inore firmly t h a n t h e fl s u b u n i t s do, a n d t h a t t h e a b n o r m a l i t y in the (~ s u b u n i t s of tile H b M molecule affects functions of the n o r m a l fl s u b u n i t s to a g r e a t e r e x t e n t t h a n the a b n o r m a l i t y in t h e fl s u b u n i t s affects those of the n o r m a l a subunits. These suggest t h a t t h e a a n d fl s u b u n i t s in t h e H b A molecule m a y be f u n c t i o n a l l y u n e q u i v a l e n t . The a b n o r m a l a s u b u n i t s of H b MBoston a n d M Iwate were more stable, b u t the a b n o r m a l ~ s u b u n i t s of H b Msaskatoon were less stable, against d e n a t u r a t i o n b y b e n z o a t e t h a n H b A. Similarly, the molecule of H b MBoston was more stable, b u t t h a t of H b Msaskatoona n d Mnyde Park were less stable, against d e n a t u r a t i o n b v heat t h a n t h a t of H b A. These i n d i c a t e t h a t the a b n o r m a l i t y in the a s u b u n i t s was responsible for reinforcing the molecule, whereas the a b n o r m a l i t y in tile/~ subunits was responsible for enfeebling t h e molecule. T h e results of t h e s t u d y of the d e n a t u r a t i o n in vitro a p p e a r to be c o m p a t i b l e with those of t h e hematologic s t u d y in vivo: those who have H b MSaskatoon a n d MI~yae Park show a p p a r e n t signs of mild h e m o l y t i c anemia, whereas those who h a v e H b MBoston a n d M Iwate show no sign of h e m o l y t i c anemia.

* This paper was read at the 7th International Congress of Biochemistry, Toky(). 19~'7¢ (ref. l). Biochim. Biophys. =~cta, I08 (I9(,8} 26,~ -'73

PROPERTIES OF HEMOGLOBIN l~I

263

INTRODUCTION

In hemoglobin researches, genetically determined abnormal hemoglobins are useful materials in order to elucidate relationships between the structure and function of H b molecules, because of their specific modifications. A m o n g various abnormal hemoglobins, hemoglobins M are especially useful, for their structural abnormalities are located in regions closely connected with their heme groups. In Figs. i a and b,

/ - A

--.......

//

/

556 8

. ...

iCys, 6

......

~' 94

Lys 65(

c

~/

/

NT

NT

Fig. i. Schematic illustrations of the a and fl subunits shown are based on the model of the a and fl subunits of CULLISet al3. (a) a subunit. Hb MB: E 7 (58) His --~ Tyr; Hb MI: F8 (87) His --~ Tyr. (b) fl subunit. Hb Ms: E 7 (63) His --~ Tyr; Hb MH : F8 (92) His --~ Tyr.

the structures around the heme groups of the a and fl subunits of a H b molecule are schematically presented. Hb MBoston(Hb Ms), H b Msaskatoon (Hb Ms), H b M Iwate (Hb M I) and H b MHyde Park (Hb MH) are known to have one of the histidine residues replaced b y tyrosine residue in each molecule: the substitutions are at E 7 * o f a subunit for Hb MB (a58) (ref. 4), at E 7 of fl subunit for H b Ms (fl63) (ref. 4), at F 8 * of a subunit for H b M I (a87) (ref. 5) and at F 8 of fl subunit for H b MH (f192) (ref. 6). The histidine residue at E 7 (distal histidine) is spatially closer to the functional site of the reversible oxygenation than t h a t at F 8. On the other hand, the histidine residue at F 8 (proximal histidine) occupies the fifth coordination position of the heme iron in a and fl subunits. Since these four hemoglobins M have the same kind of amino acid substitution, histidine with tyrosine, the substitution is doubtless homologous in effect on the heine irons of the hemoglobins ; the difference is whether the abnormality is in the a or fl subunits and in the distal or proximal histidine. This report deals with comparative s t u d y on the stability of these four hemoglobins M, and will present evidence for the existence of an inherently different nature between a and fl subunits in H b molecules.

* E 7 and F 8 refer to the numbering system presented by PERUTZ8. Biochim. Biophys. Acta, 168 (1968) 262--273

264 MATERIALS

A. H A Y A S H I

et a l .

AND METHODS

Whole blood specimens were obtained from heterozygous patients possessing Hb MB (Hb Mosaka, ref. 7), Hb Ms (Hb MI~urume, ref. 8), Hb M t and Hb MH (Hb MAkita, ref. 9)' Hemolysate was prepared as described previously x°.

Isolation of hemoglobins M Various hemoglobins M were isolated in the oxy %rm from the respective hemolysate by means of chromatography on an Amberlite CG-5o colunm. The chromatography was performed at 4-6 ° with the use of o.o5 M potassium phosphate buffer (pH 7.o). Normal Hb A present in hemolysates was coinpletely eluted out of the column with the buffer mentioned above, whereas Hb Ms and MH were eluted with o.o 7 M buffer, and Hb MI~ and M 1 were eluted with o. 5 M NaC1 solution. The resulting Hb M and Hb A were dialyzed against an appropriate buffer and the dialyzed solutions were used in the oxy or carboxy forln.

Determination of reduction rate of Hb CO M by sodium dithionite * HbCO M in o.I M potassium phosphate buffer (pH 7.o) wa~ placed in a tonometer attached to an optical cell of o.25-cm light path filled with CO gas. The reaction was started by the anaerobic addition of freshly prepared sodium dithionite in o.o5 M borax solution and then the resulting absorbance change was followed at 6oo m/.c

Determination of oxidation rate The oxidation of hemoglobins M was evaluated in three ways as follows. All experiments were performed in the same type of tonometer as mentioned above. Autooxidation of Hb02:HbO2 in o.o 4 M potassium phosphate buffer (pH 7.o) was placed into the tonometer filled with pure 02 gas. Since the affinity to 02 of Hb Me (ref. II) and MI (ref. 12) is so low that they could not be completely saturated with atmospheric oxygen, the Hb was then allowed to stand at 5 ° until the absorbance of Hb became constant (for about IO min). The absorbance change accompanying the autooxidation of Hb was measured at 37 ° over the range of wavelength from 45o to 65o m#. In some cases, the reaction was carried out in o.o4 M potassium phosphate buffer (pH 7.o) containing 2.o M NaC1 and also in o.16 M sodium acetate buffer

(pH 5.0). Oxidation of HbCO by potassiumferricvanide : HbCO in o. I M potassium phosphate buffer (pH 7.o) in the tonometer filled with pure CO gas was oxidized to MetHb by the addition of potassium ferricyanide (final concentration, 2.14" IO-2 M). The concentration of HbCO M used in this study was twice as high as that of HbCO A ; thus the concentration of HbCO M and HbCO A was the same per ferrous heme. The oxidation was started by the injection of potassium ferricyanide, and the absorbance change was then measured at 25 ° and at 568 m#. Oxidation of HbO 2 by sodium nitrite: The procedure was the same as that for assay of the oxidation by potassium ferricyanide, except that HbO~ and sodium * "['he a b b r e v i a t i o n s u s e d a r e : H b C O M , c a r b o n m o n o x i d e h e n l o g l o l ) i n M : (~2Fea4 Tyr/J2Fe"' ('O o r a2 v e ~ -CO/~2Fe~-TYr; H b O 2 M, o x y h e m o g l o b i n M : et2 Fe3+ Tyr/~2Fe~+ O~ o r 6~oFe~* O'~ /] F(,a, Tyr; M e t Hb M, methemoglobin M : a2 Fe3* Tyrfl2Fe"+-H'~O o r C~2Ve~+ H'Ofl,oFe"'-T-vr.

Biochim. Biophys, Acta, m8 (r908) 2()2 z73

PROPERTIES OF HEMOGLOBIN M

265

nitrite (final concentration, 5" lO-5 M) were used. In the oxidation of HbCO b y sodium nitrite, accurate data could not be obtained.

Determination of denaturation rate The following two methods were employed. (I) Reaction of MetHb with sodium benzoate: Sodium benzoate was added to MetHb solution (final concentration, 5" lO-5 M in o.i M potassium phosphate buffer (pH 7.o)), and the mixture allowed to stand at 25 ° for 60 rain. The resulting mixture was then assayed for absorption spectrum in the range of wavelength from 450 to 650 m#. The percentage formation of the hemichrome was calculated b y the following equation : Altb -- A obs. hemichrome(°/0) -× ioo AHb -- A hemi.

A i~b, absorbance of initial MetHb; Aobs., absorbance at a benzoate concentration ; A hemi., absorbance as all the MetHb has been converted into hemichrome. (2) Heat denaturation of MetHb: MetHb (final concentration, 6. lO -5 M in o.i M potassium phosphate buffer at p H 6.8) placed in a small test tube (diameter, 0.3 cm) was incubated for 2 rain in a water bath, the temperature of which was regulated to 5 o°, and then for an appropriate length of time in another water bath, the temperature of which regulated to 64 °, immediately followed b y cooling in an ice water bath. The cooled reaction mixture was subjected to the measurement of absorption at 500 and 630 m # after centrifugation. All spectra were measured b y a Cary model 15 recording spectrophotometer. The H b concentration was determined b y the alkali-denatured globin hemochrome method 13, assuming the eM at 559 m # to be 30.6" I o 3. RESULTS

Reduction of HbCO M by sodium dithionite When reduced b y sodium dithionite under N 2 gas for prolonged period of time, H b M changes its absorption spectrum, and the changed absorption spectrum is identical with that of deoxy Hb A (ref. 14). The reduction of H b M is much faster in rate under CO gas than under N 2 gas, resulting in the formation of HbCO M' *, the absorption spectrum of which is identical with t h a t of HbCO A (ref. 15). W h e n compared among H b Ms, H b MH and Hb MB, the reduction rates were significantly different from one another; Hb Ms was 50% reduced in 15 sec, H b MH in 55 sec and Hb MB in 260 sec (Fig. 2). Under N 2 gas, the reduction rate of Hb M i was slower than t h a t of Hb MB. The reduction rate of MetHb A to HbCO A under CO gas was too rapid to be measured with accuracy.

Oxidation of HbO 2 and Hb CO (I) Autooxidation of Hb02: The autooxidation of the normal heroes of H b M * HbCO M'. This Hb is formed from HbCO M by the action of dithionite under CO gas and its absorption spectrum is identical with that of HbCO A. The reaction may be represented by the following formula: HbCO M dithionite HbCO M" ~2Fe3+ T y r ~ 2 F e ~ + - C O

> (x2Fe2+--CO . . . . . . . . T y r ~ 2 F e * + - C O

dithionite a 2 F e 2 + - C O ~ 2 F e B + - T Y r --

> a 2 Fe~+-CO f l 2 F e i + - C O . . . . . . . .

Tyr

Biochim. Biophys. Acta, 168 (1968) 262 273

266

~. HAYASHI et al.

2 . 0 ~.7~$--. . . . . . .

2.0

"\ \ 1.5 N.N

v

"\ 0 U XS

X

~.o

F

\

o~

N

X3

'\

Xt~ 1.E

\

O

1

2

3

4

5

0

Time (mln)

1

2

"3

4

5

Time(h)

Fig. 2. R e d u c t i o n o f t l b C ( ) M b y s o d i u m d i t h i o n i t e . H b C O M (final c o n c e n t r a t i o n , 3 . 2 5 . ~o 4 M as h e n l e ) in b o r a t e p h o s p h a t e b u f f e r w a s r e d u c e d w i t h s o d i u m d i t h i o n i t e (final c o n c e n t r a t i o n , t. 5 . l o -2 M) a t p H 7.o. T h e r e a c t i o n w a s p e r f o r m e d in CO g a s a t 25 ° a n d m e a s u r e d a t 6 o 0 rap. , HbCO Mn; ..... , H b C O Ms; . . . . . , HbCO Mu. Fig. 3- A u t o o x i d a t i o n o f H b O = M a n d H b O 2 A. A u t o o x i d a t i o n o f H b O 2 (filial c o n c e n t r a t i o n , 2. 5 . ~o -~ M as h e i n e ) in o . o 4 M p o t a s s i u m p h o s p h a t e b u f f e r ( p H 7.o) w a s p e r f o r m e d in ()a g a s at 37°. The calculation was m a d e at 576 mff. O - - O , HbO,2M~;[~ ~,HbO2M1; ,: . .ll[~()2Ms; ~._~, H b O s M H ; ( ) (",, H b O s A.

was estimated under tile three different conditions; (I) at pH 7.o, (e) at pH 5.0 and (3) at a high salt concentration. In o.o4 M potassium phosphate buffer (pH 7.o) the autooxidation rates of Hb MB and M i which had the abnormality in the a subunit, were remarkably faster than those of Hb A, and Hb Ms and MH which had the abnormality in the fi subunit (Fig. 3). The time courses with all hemoglobins tested were of the first order; Hb MB, M t, Ms, MH and Hb A were 5o% oxidized in 4.1, 3-4, 3o.1, 9.4 and 18.8 h, respectively. Effects of low pH (5.o) and high salt concentration (e M NaC1) on the autooxidation were also investigated with Hb MB, Ms and Hb A. The autooxidation rates of Hb MB and Hb A increased, particularly in the presence of e M NaC1, and the increases were similar for both hemoglobins. The rate of Hb Ms, however, could not be determined under these conditions, because an abnormal reaction occurred in the early stage of the autooxidation ; the absorption spectrum of Hb Ms in the early stage of autooxidation closely resembled that of HbO 2 Ms in the presence of sodium benzoate (final concen~ ,.~:

2.0

~ •.$ ....

t~j 1.O

Z

0

\

'

1

2 :3 Time (mini

4

5

Fig. 4. O x i d a t i o n o f H b C O M a n d H b C O A b y p o t a s s i u m f e r r i c y a n i d e . H b (final c o n c e n t r a t i o n for H b C O M , 4"IO a M as h e i n e a n d for t t b C O A , 2 . i o - 4 M as h e m e ) in o.1 M p o t a s s i u m p h o s p h a t e b u f f e r ( p H 7.o) w a s o x i d i z e d w i t h p o t a s s i u m f e r r i c y a n i d e (final c o n c e n t r a t i o n , e . I 4 • IO -2 M) u n d e r CO gas. R e a c t i o n w a s m e a s u r e d a t 25 ° a n d a t 5 6 8 mff. - . , HbCO Mn; ..... , HbCO Ms; ...... , HbCOMH; - - -- , H b C O A .

Biochim. Biophys..4cta, i 6 8 (1008) 2 6 2 -'73

267

PROPERTIES OF HEMOGLOBIN M

tration, 4" 1°-1 M), and the absorption spectrum of the product b y the complete autooxidation was entirely different from that of MetHb Ms. This abnormal reaction of H b Ms m a y be related to a denaturation. (2) Oxidation of HbCO by potassium ferricyanide: The time courses of the oxidation of HbCO by ferricyanide were of the first order. As shown in Fig. 4, Hb MB was most rapidly oxidized; it was 50% oxidized in only 5 sec. The oxidation rates of l i b Ms and MH were faster than that of H b A ; H b Ms, MH and H b A were 50% oxidized in 9 o, 7 ° and 165 sec, respectively. (3) Oxidation of Hb02 by sodium nitrite: The oxidation of HbO~ b y nitrite was remarkably different in curve for time course from the autooxidation and from the oxidation by ferricyanide; the curves for the oxidation of HbO 2 by nitrite were of sigmoid shape but not of first order (Fig. 5). Hb MB, Ms and Hb A were 50% oxidized in 48, lO2 and 244 sec, respectively. It m a y be interesting to note that difference in oxidation by nitrite between Hb MB and Ms was not so remarkable as the difference in autooxidation (Fig. 3).

\

\

\ \

-~ ao

\

\

,

1

~,

.

2

"~.

\

.

3

Time (rain)

4

,-.. 5

,~ 6

Fig. 5. O x i d a t i o n o f H b O ~ M a n d HbO~ A b y s o d i u m n i t r i t e . HbO~ (final c o n c e n t r a t i o n for H b O 2 M, 4.2. lO -4 M as h e m e a n d for H b O 2 A, 2.1. lO -4 M as heine) in o.i M p o t a s s i u m p h o s p h a t e buffer (pH 7.0) w a s o x i d i z e d w i t h s o d i u m n i t r i t e (final c o n c e n t r a t i o n , 5 " i o-* M). R e a c t i o n w a s p e r f o r m e d in 02 gas a t 25 ° a n d m e a s u r e d a t 576 m # . , H b O , MB; . . . . , H b O 2 Ms; - - , HbO2 A.

Denaturation of MetHb (i) Reaction of MetHb with sodium benzoate: MetHb A, when mixed with benzoate, forms the hemichrome. The absorption spectrum of MetHb A and that of the hemichrome have an isosbestic point at 596 m#. At benzoate concentrations higher than 1. 5 M, the absorption spectra of Hb MB, M i and Ms became indistinguishable from that of the hemichrome of MetHb A. Since at various benzoate concentrations the spectral change of the normal subunits of methemoglobins M is expected to be identical with that of MetHb A, it is conceivable that changes in absorbance at 596 m# with methemoglobins M, if observed, are due to their abnormal subunits. MetHb A was 50% converted into the hemichrome at 0.59 M benzoate (Fig. 6). On the other hand, higher concentrations of benzoate were required for the abnormal a subunits of Biochim. Biophys. Acta, 168 (1968) 262-273

A. HAYASHIel al.

268

27

100

/

....

/ 1.%

o%

"\..

7

e 50 8 o 6

1.O -%0

-0.5

6

log [sodium benzoate (M)]

Q5

r;,

1

2 13 Time (rain)

a

5

Fig. 6. R e a c t i o n of t h e a b n o r m a l s u b u n i t s o f M e t H b M a n d M e t H b A (final c o n c e n t r a t i o n 5 " I o - 5 M a s h e m e ) i n o . l M p o t a s s i u m p h o s p h a t e b u f f e r ( p H 7.o) w i t h s o d i u m b e n z o a t e w a s p e r f o r m e d a t 25 °. C a l c u l a t i o n w a s m a d e a t 596 ml* for m e t h e m o g l o b i n s M a n d 536 m y for M e t H b A (refer t o t h e t e x t ) , q ~ - - O , M e t H b MB; × - - - x , M e t H b Ms; [~---[~, M e t H b MI; <)- (3, M e t H b A. Fig. 7. H e a t d e n a t u r a t i o n of M e t H b M a n d M e t H b A, M e t H b M a n d M e t H b A (final c o n c e n t r a t i o n , 6. i o -s M) i n o . t M p o t a s s i u m p h o s p h a t e b u f f e r ( p H 0.8) w e r e i n c u b a t e d a t 04 °. M e a s u r e i n e n t of t h e r e m a i n i n g M e t t t b w a s m a d e a t 5oo a n d 63 ° m r . O - - O , M e t H b M n ; -" - > . M e t H b Ms ; A /~, M e t H b MH; (-) ~ , M e t H b A.

Hb M B and M i to be converted into hemichromes; they were 50/o o/ converted into the henfichromes at o.68 and 0.85 M benzoate, respectively. The spectral change at 596 m# of MetHb Ms, however, occurred at considerably lower concentrations of benzoate than those of MetHb M B and MI; MetHb M s was 5 ° % converted into the hemichrome at o.33 M benzoate. (2) Heal denaturation of MetHb: When heated at 64 °, n~ethemoglobins were denatured (Fig. 7)- Doubtless, slight lag phases at the initial stage of the denaturation resulted from the time required for raising the temperature of the reaction mixture from 5°0 to 64 ° . Time courses of the denaturation rates were of the first order except for those at the initial stage. In the heat treatment, MetHb MB was more stable, but MetHb Ms and M~I were less stable than MetHb A; MetHb MB, Ms, Me and A were 50% denatured in 14. 9, i. 9, 1.6 and 4.1 rain, respectively. DISCUSSION

The heroes of the Hb molecule play an important role not only as a functional site but also as a stabilizer of the molecule. The variants of Hb M used in the present study were Hb MB, M I, Ms and MI-I, in all of which tyrosine is substituted for the histidine residue that is locating near the heme in the normal Hb molecule (Fig. I a and b). The results obtained in the present and preceding studies are summarized in Table I. Advantages for the use of hemoglobins M may be that properties of the a and fl subunits of Hb molecule can be analyzed as they are in the molecule.

Reactions of hemoglobins M with ligands In abnormal subunits: It has been generally accepted that in the abnormal subunits of hemoglobins M, the substituted tyrosine residue forms a stable bonding, so-called an intramolecular bonding, with the ferric heine iron*. In the reaction of Biochim. Biophys. Acla, t 6 8 ( i 9 6 8 ) 262 273

"x3

o

03

TABLE I

©

COMPARISON OF THE PttYSICOCHEMICAL PROPERTIES OF Tltl~ FOUR VARIANTS OF H b M * A b n o r m a l s u b u n i t ; "~ n o t e x a m i n e d ; ***, p r e s e n t s t u d y ; - - < H b A; i

Physicochemical properties

a S u b u n i t abnormality Hb MB (distal) at

R e d u c t i o n r a t e by d i t h i o n i t e Affinity for c y a n i d e a n d azide Affinity for fluoride Affinity for s o d i u m b e n z o a t e Chartge of E P R s p e c t r a of t h e a b n o r m a l s n b u n i t i n d u c e d d u r i n g o x y g e n a t i o n of t h e n o r m a l s u b u n i t A u t o o x i d a t i o n r a t e in 0 2 f o r m O x i d a t i o n r a t e b y f e r r i c y a n i d e in CO f o r m O x i d a t i o n r a t e b y n i t r i t e in 0 2 f o r m Affinity for O~

± ±

t~

A b o v e p H 7.5 a s l i g h t B o h r effect w a s observed1%

Hb 3 I i ( p r o x i m a l )

H b M s (distal)

+++ + + + +

++++

©

. . . . . . . .

~ ~ ~

± ~ ±

± ++ slight

++++ ** **

:z

H b 2~Ilt (proximal) a

**

120

Denaturation by heat E l u t i o n r a t e in c o l u m n c h r o m a t o g r a p h y

© S u b u n i t abnormality

fl

±

Effect o f p H on 0 2 affinity (Bohr effect)

o0

~ H b A ; + > H b A.

N

fl*

4-

Io

IO

remarkab le

i6

+ ++

-

+ ++

+

II.

12

17, 18 ------3

4-or+

±

++

or+

II,

I2

17 , 18 +++

27 °

A. HAYASHI et al.

HbCO M with sodium dithionite (Fig. 2), it seems likely t h a t the intramolecular bonding is so stable that the heine iron is reduced at a slow rate b y the powerful reducing agent, dithionite. The fact that the reductions of HbCO MI and M B are slower in rate than those of HbCO MH and Ms, is possibly due to that the former and the latter carbon monoxide hemoglobins M are different in stability of the intra nlolecular bonding. The rates of reactions of the abnornlal subunits of hemoglobins M with cyanide and with azide are in parallel to the rates of reduction of carbon monoxide hemoglobins M b y dithionite ; Ht) M i and MB have lower affinity to cyanide and azide than H b Ms has ~°. Conceivably, tile intranlolecular bonding is significantly more stable in the a subunits than in 'tthe fi subunits of Hb molecules and is more stable in the 5th than tile 6th coordination position of each of the subunits. In normal subunits : Either in the oxidation of HbO 2 M by molecular (~xygen or in the oxidation of HbCO M by ferricyanide, the rates were remarkably higher with H b MB and M I, abnormal in the a subunits, than with H b Ms and MH, abnormal in the /3 subunits (Figs. 3 and 4). Earlier, BETKE and his associates suggested that the ferrous irons of the H b molecule are protected by tile bound ()2 ()r CO against attacks by other oxidizing ligands, and that the rates of tile attacks are dependent on the binding strength of the ligands~", 2°. The difference in oxidation rate among hemoglobins M mentioned above mav be explained as well; the normal ~, subunits of H b Ms and MH are capable of binding with O 2 and with CO more firmly than the normal [3 subunits of Hb MB and MI. The previous studies on the O2 equilibrium of hemoglobins M have revealed t h a t in affinity to O2, H b MB (ref. I I ) and MI (ref. 12) are significantly lower than H b A, whereas Ht) Ms (ref. 17) and MH (ref. i8) are almost tile same as HI) A. It seems likely, therefore, t h a t in both abnormal and normal subunits of henloglobins M, the a heme iron can bind ligands more firmly than the/3 heine iron. The difference in strength of the ligand heine bonding suggests that the distance fronl the heine iron to the proxilnal or distal histidine residue is smaller in the a than in the /3 subunits.

Interaction between subunits of hemoglobins M There is an alternative explanation for the functional abnormalities of Hb M In the case of hemoglobins M having ferric heroes in the abnormal subunits and ferrous ones in the normal subunits: In tile oxidations b y molecular oxygen and by ferricyanide (Figs. 3 and 4) as well as in the affinity to 02, the nornlal ferrous fl subunits of Hb MB and Ml showed remarkable abnormalities in function (refs. i i and i2), whereas the normal ferrous a subunits of H b Ms and MH were functionally similar to H b A (refs. 17 and 18). Seemingly, there is some interaction between the a and tile /3 subunit, but the strength of the interaction is different of the henloglobins M ; the interaction between the abnormal ferric a subunit and the normal ferrous/3 subunit is stronger than t h a t between the abnormal ferric/5 subunit and tile normal ferrous a subunit. The normal fl subunits of H b MB and MI m a y have the molecular conformation distorted by such a strong interaction, thus showing the functional abnormalities. ]'he difference in chronlatographic behavior of oxyhemoglobins M on CG-5 o m a y also reflect this molecular distortion ; H b MB and H b MI were markedly slower in tile elution rate than H b Ms and MH were, the rates of which were rather resembled to t h a t of H b A. It was previously reported t h a t H b Ms (ref. 17) and MH (ref. IS) show tile Bohr Biochim. Biophys. ~4cta, t08 (t968) 262 273

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effect, but Hb MB (ref. II) and MI (ref. 12) do not. It was reported also that the electron paramagnetic resonance (EPR) spectrum of deoxy Hb MH is remarkably different from that of Hb02 MH (ref. 16). These findings indicate that Hb Ms and MH, when oxygenated, change their conformations, especially of the abnormal/5 subunits, which can not combine with 03; in Hb MH, the electronic state of the ferric hemes of the abnormal/5 subunits is remarkably affected by the oxygenation of the ferrous hemes of the normal a subunits. On the other hand, there are little evidences whether Hb MB and MI exhibit such a conformation change associated with oxygenation of the normal /5 subunits. In the case of hemoglobins M having ferric heroes in both normal and abnormal subunits: HAYASHIand his associates 1° found that when the heine irons of both normal and abnormal subunits of hemoglobins M are in the ferric state, the normal fl subunits of Hb MB and MI do not show the abnormalities mentioned above to a significant extent ; the affinity of the/5 subunits of the hemoglobins M towards cyanide, azide and fluoride are similar to those of Hb A. This finding suggests that the fl subunits of Hb MB and MI resemble those of Hb A with respect to both structure and function, when they are in the ferric state. In the previous report ~1, however, it was also suggested that the/5 subunits of MetHb MB can exhibit a slight distortion under some conditions; the E P R spectrum of the azide complex of the/5 subunits of Hb MB is slightly different in g-values from that of the azide complex of Hb A, whereas the E P R spectrum of Hb Ms is similar to that of the azide complex of Hb A.

Relationship between structure and function of hemoglobins M and Hb A The most prominent features of hemoglobins M from other abnormal hemoglobins and Hb A may be as follows: (a) The presence of the intramolecular bonding between the substituted tyrosine residues and the ferric heine irons; the difference in property of the intramolecular bondings of the hemoglobins M may be due to difference in structure around the heroes of the abnormal subunits. (b) Hemoglobins M are a kind of the hybrid molecule, composed of two normal subunits carrying ferrous hemes and two abnormal subunits carrying ferric ones; when the hemes are in such states, the abnormal subunits produce a powerful effect on the normal subunits, and thus result in structural distortion of the latter subunits, especially in the case of Hb MB and MI. On the other hand, when the hemes of both subunits are in the ferric form, the structural distortion of the normal subunits is not so significant. Many investigators have studied the structure and function of Hb moleculeZ*,~3, particularly the functional roles of the a and fl subunits, from experimental and theoretical point of view .4-26. It is not yet clarified, nevertheless, whether the a and the fl subunits in Hb molecules are functionally equivalent or not. Recent studies with the subunits isolated from the Hb molecule have not been successful to distinguish the roles of the a and the /5 subunits2~, *s. These negative findings, however, may suggest that the difference in intrinsic nature of the a and t5 subunits, if it is, is very small. A significant difference in physicochemical properties between the Hb MB and MI having the abnormal a subunits and the Hb Ms and MH having the abnormal fl subunits (Table I) strongly suggests that the a and the t5 subunits have unequivalent Biochim. Biophys. Acta, 168 (1968) 262-273

272

A. HAYASH1 el a[.

nature in Hb molecule, though this unequivalency may vary in extent by a specific character of Hb M.

Denaturation of hemoglobins M It was shown that tile abnormal a subunits of Hb MB and Mi were more stable against denaturation by benzoate than Hb A and the abnormal [3 subunits of Hb Ms (Fig. 6). Also in heat denaturation, Hb MB was more stable than Hb A and Hb Ms and MH (Fig. 7). It seems likely, therefore, that a stable intramolecular bonding in the abnormal ~t subunits of Hb MB and MI is responsible for the high stability of the whole molecule. This is in a good accordance with STEINHARDT and his associates "~, who have suggested that the firmness of the bonding between ligand and heme ir(m in the Hb molecule is closely related to the stability of the Hb molecule. On the other hand, the intramolecular bonding in the/5 subunits can contribute only slightly to stabilization of the molecule: Hb Ms is more stable than Hbz(iricha°,al which has arginine substituted for the same histidine residue as for Hb Ms, though both the abnormal hemoglobins are less stable than HI) A is. The facts, that the abnormal fi subunits of Hb Ms and MH are faster in rate of the reduction by dithionite (Fig. 2) and higher in affinity to cyanide and azide than the abnormal (l subunits of Hb M~ and MI (ref. 9), suggest that the strength of the intramolecutar bonding is less stable in the [4 than in the ~ subunits. But the instability of the [3 subunits mentioned above is not explainable merely by the strength of the intramolecular bonding, because Hb A without such an intramolecular bonding is more stable than Hb Ms and MH with it are. BUNg ANI) JANI)La~ and ]~ANEI{JEF- AND CASSOI.Yaa suggested that in the Hb A molecule the heme globin linkage of the [3 subunits is also less stable than that of the ~t subunits. It is assumed, therefore, that in the normal Hb A molecule the/J subunits are not so rigid in structure that they exhibit the confi)rmation change associated with oxygenation. But when substitution of an amino acid occurs in the/:1 subunits the molecule is easily denatured, although the extent of denaturation differs depending on kind of the substituted amino acid and location of the substitution. It was previously reported that patients producing Hb Ms (retls. 34, 35) and MH (ref. 9) show signs of mild llemolyt:ic anemia; in patient producing Hb MB, the red cell life span by the La°-P]DFP lnethod was 137 days and one-half the liik~ span by the 5~Cr method was 29 days. The results of denaturation in vitro study appears compatihle with those of i~ vivo hematologic study. So far as reported, most of the hemoglobinopathies accompanied by hemolytic anemia have an atmormalitv in the fl subunits but not in the ~ subunits of the Hb molecule.

ACKNOWLEDGEMENTS

We express our gratitude to Dr. T. HoRIo for his general interest in the woblem and helpful comments, and to Dr. I. T~'UMA, Dr. H. WATARI and Dr. H. MORIMOTO for valuable discussion. We also wish to thank Dr. S. SHIBATAfor the blood specinlen of the patient with Hb M~yde Park (Akita), Dr. N. KIMURA for the specimen with H b Msaskatoon (gurume) and the late Dr. A. TAMURAfor the specimen with Hb MIwate.

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REFERENCES i A. HAYASHI, A. SHIMIZU, T. SUZUKI AND Y. YAMAMURA, Proc. 7th Intern. Congr. Biochem., Tokyo, 1967, A b s t r a c t s 5, The Science Council of J a p a n , Tokyo, 1967, p. lO42. 2 A. V. CULLIS, H. MUIRHEAD, M, F. PERUTZ, M. G. ROSSMANN AND A. C. T. NORTH, Proc. Roy. Soc., London, Ser. A, 265 (1962) 161. 3 M. F. PERUTZ, J. Mol. Biol., 13 (1965) 646. 4 P. S. GERALD AND M. L. EFRON, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1758. 5 S. SHIBATA, A. TAMURA, I. I u c m AND T. MIYAJI, Proc. Japan Acad., 4 ° (1964) 220. 6 P. HELLER, R. D. COLEMAN AND V. J. YAKULIS, J. Clin. Invest., 45 (1966) lO21. 7 A. SHIMIZU, A. HAYASHI, Y. YAMAMURA, A. TSUGITA AND I{. KITAYAMA, Biochim. Biophys. Acta, 97 (1965) 472. 8 S. SHIBATA, T. MIYAJI, I. IUCHI, S. UEDA, I. TAKEDA, N. KIMURA AND S. KODAMA, Nippon Ketsueki Gakkai Zasshi, 25 (1962) 690. (in English). 9 S. SHIBATA, T. MIYAJI, K. KARITA, I. IUCHI, Y. OHBA AND K. YAMAMOTO, Proc. Japan Acad., 43 (1967) 65. IO A. HAYASHI, A. SHIMIZU, T. SUZUKI AND Y. YAMAMURA, Biochim. Biophys. Acta, 14o (1967) 251. I i T. SuzuKI, A. HAYASHI, Y. YAMAMURA, Y. ENOKI AND I. TYUMA, Biochem. Biophys. Res. Commun., 19 (1965) 691. 12 N. HAYASHI, Y. MOTOKAWA AND G. KIKUCHI, J. Biol. Chem., 241 (1966) 79. 13 M. SUZUKI, A. I~AJITA AND C. I~IANAOKA,J. Biochem. Tokyo, 187 (195 o) 393. 14 Y. MOTOKAWA, N. HAYASHI AND G. KIKUCHI, Arch. Biochem. Biophys., lO 5 (1964) 612. 15 P. S. GERALD AND P. GEORGE, Science, 129 (1959) 393. i6 A. HAYASHI, T. SUZUKI, A. SHIMIZU, H. MORIMOTO AND t{. WATARI, Biochim. Biophys, Acta, 147 (1967) 407 • 17 T. SUZUK1, A. I-IAYASHI, A. SHIMIZU AND Y. YAMAMURA, Biochim. Biophys. Acta, 127 (1966) 280. I8 A. HAYASHI, T. SUZUKI, A. SHIMIZU, K. IMAI, H. MORIMOTO, T. MIYAJI AND S. SHIBATA, Arch. Biochem. Biophys., 125 (1968) 895. 19 K. BETKE, Klin. Wochschr., 31 (1953) 573. 20 K. BETKE, ][. GREINACHER ANn V. HECKER, Arch. Exptl. Pathot. Pharmakol., 229 (1956) 207. 21 H. WATARI, A. HAYASHI, H. MORIMOTO AND M. KOTANI, in S. FUJIWARA AND L. H. PIETTE, Recent Developments of Magnetic Resonance in Biological System, Hirokawa, Tokyo, 1968, p. 128. 22 A. ROSSI-FANELLI, E. ANTONINI AND A. CAPUTO, in C. B. ANFINSEN, JR., M . L . ANSON, J. T. EDSALL AND F. M. RICHARDS, Advances in Protein Chemistry, Vol. 19, Academic Press, New York and London, 1964, p. 73. 23 A. RIGGS, Physiol. Rev., 45 (1965) 619. 24 D. E. KOSHLAND, JR., G. NEMETHY AND D. FILMER, Biochemistry, 5 (1966) 365 • 25 J. WYMAN, J. Am. Chem. Soc., 89 (1967) 22o2. 26 G. GUIDOTTI, J. Biol. Chem., 242 (1967) 3673, 3685, 3694 and 37o4 . 27 E. ANTONINI, E. BOCCI, C. FRONTICELLI, J. WYMAN AND A. ROSSI-FANELLI, J. Mol. Biol., 12 (1965) 375. 28 I. TYUMA, R. E. BENESCH AND R. BENESCH, Biochemistry, 5 (1966) 2957. 29 J. STEINHARDT, R. ONA-PASCUAL, S. BEICHOK AND CHIEN HO, Biochemistry, 2 (1963) 256. 3 ° P. G. FRICK, W. H. HITZIG AND I~. BETKE, Blood, 20 (1962) 261. 31 F. BACHMANN AND H. R. MARTI, Blood, 20 (1962) 272. 32 H. F. BUNN AND J. H. JANDL, J. Biol. Chem., 243 (1968) 465 . 33 R. BANERJEE AND R. CASSOLY, Biochim. Biophys. Acta, 133 (1967) 545. 34 A. M. JOSEPHSON, H . G . WEINSTEIN, g . J. YAKULIS, L. SINGER AND P. HELLER, J. Lab. Clin. Med., 59 (I962)[918. 35 N. HOBOLTH, Acta Paediat. Scand., 54 (1965) 357.

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