Kinetics and mechanism of hemoglobin denaturation in alkali

86

Biochimica et Biophysica Acta 830 (1985) 86-94

Elsevier BBA32252

Kinetics and mechanism of hemoglobin denaturation in alkali

Douglas R. Wilson and A.H. Burr * Department of Biological Sciences, Simon Fraser University, Burnaby, B.C., V5A IS6 (Canada)

(Received December 17th, 1984) (Revised manuscript received April 17th. 1985)

Key words: Hemoglobindenaturation; Chemical modification; Sulfhydryl group; Superoxide; (Human) The denaturation of oxy, deoxy, CO and met derivatives of human hemoglobin A at pH 11.7 and 25°C was followed by three assay methods: chromophore absorbance (which indicates changes in the heme), and the amount of precipitation in 24% ammonium sulfate neutral buffer or 0.1 M NaCI neutral buffer (which indicates degree of destabilization of the protein structure). We find that oxyhemogiobin denatures in two parallel reaction sequences. The rate of sequence I is increased when the sulfhydryl groups in the cq[3~ subunit interface have been modified by binding p-hydroxymercuribenzoate (which forces monomer formation) and is decreased by the binding of - H g O H (which, unlike the sulfhydryls, is not ionized at pH 11.7). These results support a mechanism in which the net rate of monomer formation is rate limiting and is enhanced in alkali by the ionization of sulfhydryls in the a l f l I subunit interface. In subsequent rapid reactions, ferric hemoglobin and Iow-salt-precipitable protein are formed. The formation of an oxidant, such as superoxide, is indicated by the kinetics of suifhydryl oxidation. The same oxidant would be available to initiate the second sequence by oxidizing some of the unreacted oxyhemoglobin to methemoglobin. During methemoglobin denaturation, as in sequence II, a low-salt-soluble ferric hemochrome is formed. In both reactions, this intermediate becomes low-salt-precipitable at the same rate. Deoxyhemoglobin denatures to ferrous hemochrome at the same rate as oxyhemoglobin denaturation in sequence I, and provided oxygen is excluded, the denaturation is fully reversible on neutralization. In the absence of oxygen, CO-hemoglobin does not denature to any detectable extent. The destabilization of hemoglobin structure that was indicated by precipitability occurred only for ferriheme derivatives and was independent of disulfide formation.

Introduction Because the structure of human hemoglobin A is known in detail, a study of its denaturation can provide insight into how proteins are held together in their native configuration. Denaturation in alkali is of particular interest because of the influence that ionization of intrinsic sulfhydryl and hydroxyphenyl groups, or neutralization of amino groups, may have on stability. However, compared to denaturation in acid or neutral conditions [1-5], * To whom correspondence should be addressed.

the mechanism of alkaline denaturation of hemoglobin has been little studied. At pH 11, human hemoglobin A is fully dissociated into aft dimers [6-9], which denature at higher pH. The denaturation rates of hemoglobin A, fetal hemoglobin and bovine hemoglobin are correlated with the different numbers of sulfhydryl groups located in the alfll interface [10,11]. Guidotti et al. [12] predicted that the rates of both alkaline denaturation and subunit hybridization should depend on the rate of monomer formation, and Perutz [13] postulated that the rate may be increased by the ionization of the a 1fll sulfhydryls.

0167-4838/85/$03.30 '.~:~1985 ElsevierScience Publishers B.V. (Biomedical Division)

87 We provide experimental support for both hypotheses. In addition we find a second, parallel, reaction sequence for oxyhemoglobin. Comparison of the denaturation reactions of oxy-, deoxy, COand methemoglobin provides further details. Our observation that CO-hemoglobin is stable in alkali is contrary to the results of previous studies in which oxygen was not carefully excluded. Methods and Materials

Preparation of hemoglobin and derivatives Hemolysates were prepared by a modification of the method of Oison [14]. Fully purified hemoglobin A, prepared by the method of Williams and Tsay [15], was used where kinetic parameters were measured. In either case, the hemoglobin was transferred to unbuffered 0.10 M NaC1 by gel filtration. Freshly prepared samples were always found to be free of ferric Hb by a method which can detect 2% ferric Hb [16]. DeoxyHb was prepared in the reaction vessel by purging H b O 2 with C O S r e e nitrogen and reacting with less than 0.2 m g / m l solid Na2S204 (dithionite). Residual dithionite in the reaction mixture ensured that oxygen and oxidants were absent. H b C O was prepared by purging with 2% CO in nitrogen, also CO2-free. Ferric Hb was prepared by reacting Tris-HCl-buffered H b O 2 A with a 2-fold excess of potassium ferricyanide for 30 min, then equilibrating with 0.10 M NaCI by gel filtration. Mercurated hemoglobins were prepared just prior to use by diluting H b O 2 A or aquometHb with 0.10 M NaCI containing the mercurial, and allowing time for complete reaction.

Alkaline denaturation experiments The denaturation reaction was initiated by abruptly adding 10.0 ml of alkaline buffer (50.0 m M Na3PO4/0.100 M NaC1 (pH 12.0)) to 10.0 ml of 0.10 m M H b O 2 A in 0.10 M NaC1. This raised the p H to 11.7 (Fisher Microprobe combination electrode, range 0 - 1 4 pH, and Corning Model 12 p H meter). The temperature was 25.0_+0.1°C. Because of the sensitivity of the denaturation kinetics to pH, the alkaline buffer components were measured to 0.02% precision. In two of the assays for extent of denaturation,

1-ml aliquots of the reaction mixture were tested for the amount of protein that precipitates when added to 4 ml neutralizing buffer. After 2 - 4 h at pH 6.8, aggregated protein was removed by centrifugation at 35000 × g for 30 min. The remaining soluble protein was determined at 275 nm. Absorbance values were expressed as fractions of the 'initial' absorbance, the absorbance of a mixture of 4.00 ml of neutralizing buffer, 0.500 ml of the alkaline buffer and 0.500 ml of the hemoglobin solution. For the 'low-salt-precipitability' assay, the neutralizing buffer was 25.0 mM sodium phosphate/0.100 M NaC1 (pH 6.4). For the 'high-salt assay' the buffer also contained 300 g / l of reagent grade (NH4)2SO 4. Untreated H b O 2 is soluble in both buffers. In the 'chromophore absorbance assay' the change in the visible spectrum was followed at 576 nm and 25°C. The fraction of native hemoglobin remaining at a particular time was calculated as F = ( A , - A f ) / ( A i - A f ) w h e r e A t, Af and A i are the absorbance at time t, final absorbance and initial absorbance, respectively.

Determination of sulfhydryl oxidation At regular time intervals, aliquots of the alkaline reaction mixture were added to neutralizing buffer containing 2.5% ( w / v ) sodium dodecyl sulfate (SDS). Alternate tubes contained 0.15 mM p-hydroxymercuribenzoate. The difference between the two 250 nm absorbance vs. time curves, minus the absorbance of an equivalent hemoglobin-free p-hydroxymercuribenzoate solution, was converted to sulfhydryls remaining per hemoglobin tetramer, using a molar extinction coefficient of 7.6- 10 - 4 M -1 • cm 1 [17]. To demonstrate the presence of disulfides, 0.050 to 0.20 ml samples were neutralized with buffer containing 2.5% SDS and 0.15 mM p-hydroxymercuribenzoate and applied immediately to 10% polyacrylamide gels. The mercurial prevented further disulfide formation.

Data analysis and precision The denaturation data were fitted by APL programs to first or second-order models using a rapidly-converging algorithm based on a non-linear least-squares procedure [18]. Terms were added to the fit model only if the resulting decrease in the fit sum of squares was assessed to be

88 s i g n i f i c a n t b y a p a r t i a l F - t e s t [19]. In the tables, R 2, the c o e f f i c i e n t o f d e t e r m i n a tion o f the fit, r e p r e s e n t s the f r a c t i o n of the o v e r a l l v a r i a n c e of the d a t a that is a c c o u n t e d for b y the fit model. I n f e r e n c e tests r e p o r t e d in the R e s u l t s w e r e b a s e d on 95% c o n f i d e n c e r a n g e s o f the fit p a r a m e ters. F o r l o w - s a l t assay d a t a f r o m f o u r o x y h e m o g l o b i n c o n t r o l s , the r a n g e s for k a, k2, B 0, B t and B 2 were +19%, +20%, +4.8%, +5.8% and + 1 0 . 4 % r e s p e c t i v e l y . F o r the h i g h - s a l t assays of t h r e e o x y h e m o g l o b i n e x p e r i m e n t s , the 95% c o n f i d e n c e r a n g e was + 20%.

Results Denaturation o f oxyhemoglobin W i t h i n 20 m i n , the n a t i v e a b s o r b a n c e s p e c t r u m is r e p l a c e d b y t h a t of a m i x t u r e of f e r r i h e m e p r o d u c t s i n c l u d i n g ferric h e m o c h r o m e (Fig. 1, inset). T h e k i n e t i c s of the a b s o r b a n c e d e c r e a s e at 576 n m are f i r s t - o r d e r w i t h a h a l f - t i m e of a b o u t 2 m i n (Fig. 1, c u r v e A ; T a b l e I, r o w 1). H i g h - s a l t s o l u b l e p r o t e i n d i s a p p e a r e d w i t h the s a m e k i n e t i c s (Fig. 1, c u r v e B; T a b l e I, r o w 2).

T h e a m o u n t of l o w - s a l t - s o l u b l e p r o t e i n dec r e a s e d w i t h two d i s t i n c t k i n e t i c c o m p o n e n t s (Fig. 1, c u r v e C; T a b l e I, r o w 3); the t w o - c o m p o n e n t m o d e l is s i g n i f i c a n t l y b e t t e r t h a n a o n e - c o m p o n e n t m o d e l ( p a r t i a l F-test, P < 0.0001). A n i n d u c t i o n p e r i o d of a b o u t 1 m i n a n d a r e p r o d u c i b l e final level of s o l u b l e p r o t e i n , always o b s e r v e d w i t h the l o w - s a l t assay m e t h o d , are e x p l a i n e d if a m i n i m u m c o n c e n t r a t i o n of u n f o l d e d p r o t e i n is r e q u i r e d before an i r r e v e r s i b l e a g g r e g a t i o n a n d p r e c i p i t a t i o n c a n occur. T h e first kinetic c o m p o n e n t has the s a m e rate c o n s t a n t as the d i s a p p e a r a n c e of H b O 2 abs o r b a n c e a n d the f o r m a t i o n of h i g h - s a l t - p r e c i p i t a ble p r o t e i n (Fig. 1; T a b l e I). W e c o n c l u d e that f o r m a t i o n o f this first l o w - s a l t - p r e c i p i t a b l e p r o d uct a n d the d i s a p p e a r a n c e of H b O 2 a b s o r b a n c e is g o v e r n e d by the s a m e r a t e - d e t e r m i n i n g step. T h e s e results i n d i c a t e that the a l k a l i n e r e a c t i o n m i x t u r e , a f t e r 20 min, is a s o l u t i o n o f two d e n a t u r e d p r o t e i n s : a l o w - s a l t - p r e c i p i t a b l e p r o t e i n that is f o r m e d d u r i n g the faster re'action, a n d a lowsalt-soluble, h i g h - s a l t - p r e c i p i t a b l e p r o t e i n that is c o n v e r t e d to a l o w - s a l t - p r e c i p i t a b l e f o r m in the s l o w e r reaction.

TABLE I OXYHEMOGLOBIN - COMPARISON OF THREE DENATURATION ASSAY METHODS AND CONCURRENT SULFHYDRYL OXIDATION Assay method and applied model

Linear parameters

Non-linear parameters a.b ( × 103 s- 1)

Fit statistics ~

DF 0

Chromophore absorbance B 1 exp( - kit )

B 1 = 0.9166+0.0022

k 1 = 6.023+0.020 (t I = 1.918 min)

S.D. = 0.00200 R 2 = 0.9999160

14

High-salt solubility B 1 exp(- kit )

B 1 = 0.949 + 0.012

k 1 = 5.67 + 0.11 (t 1 = 2.039 min)

S.D. = 0.0101 R 2 = 0.99862

10

Low-salt solubility e B0 + B 1 exp( - kit)+ B 2 exp(- k2t )

Bo = 0.2202 _+0.0030 B 1 = 0.953+0.025 B2 = 0.1300_+0.0065

k 1 = 7.08 + 0.27 (t~ = 1.63 min) k 2 = 0.240_+0.033 (t 2 = 48.0 rain)

S.D. = 0.00947 R 2 = 0.99773

35

Sulfhydryl titer B 1 exp( - kat)+ B 2 exp( - k2t )

B 1 = 2.14-t-0.12 B2 = 4.125 _+0.051

k 1 = 4.05 _+0.47 ( / 1 = 2.85 min) k 2 = 0.016_+0.001 02 = 721 rain)

S.D. = 0.108 R 2 = 0.99092

20

a

a Range given is _+1 S.D. (derived from the fit error matrix). b kn are first order rate constants in s 1; t n a r e half-times. c S.D., standard deviation of fit; R 2 coefficient of determination (multiple correlation coefficient). a Degrees of freedom. Data at times of 1 rain or less were omitted prior to fitting in order to avoid the complication of an induction period (see text).

89

\

lSO~

q o_ =

5o:

=< 500

525 550 575 WAVELENGTH•NMJ

600

625

~O4" CO

oo

oT~, o

s~ , , o

5o ~oo

soo

TIME )MINUTES)

Fig. 1. Denaturation of HbO 2 - comparison of three assay methods. A semilog plot is used in order to clearly display data covering 3 orders of magnitude in time. A. Chromophore absorbance assay (O). The absorbance change in the reaction mixture at 576 n m is plotted as the fraction of the total change vs. the time of measurement. B. High-salt assay ( x ) . The protein remaining soluble after neutralization of an aliquot of the reaction mixture with high-salt buffer is plotted as the fraction of the initial 275 n m absorbance vs. time of neutralization. C. Low salt assay ( + ). Same as B, but neutralization was with low salt buffer. The solid lines are plots of the leastsquares-fitted first-order models of Table I. The broken lines are the two exponential components that, added together, fit the low-salt assay data. Inset. Spectra of denatured and undenatured HbO 2. Solid line, absorbance spectra of reaction mixture at 20 min. Molar extinction coefficients are based on initial heme concentration. Broken line, undenatured HbO 2 in 0.10 M NaCI at p H 7.6.

Denaturation of deoxyhemoglobin DeoxyHb fully converted to ferrous hemochrome within 15 min. On neutralization with low-salt buffer in the absence of oxygen, no precipitation occurred and the absorption spectrum returned to that of deoxyHb. Subsequent oxygenation changed the spectrum to that of untreated HbO2. When aliquots were neutralized with air-equilibrated low-salt buffer, however, only partial renaturation occurred (Fig. 2). The absorption spectrum of the supernatant was indistinguishable from that of untreated H b O 2. Evidently the renaturation of ferrous hemochrome, if oxygen is present, occurs in competition with an irreversible further denaturation and precipitation. The latter provided an assay for the amount of unfolded protein in alkali. The rate constant for formation of low-salt-precipitable protein was not

oo

r

~

-

,

i

. . . . . . . . . . . TIME

r

2o

so~

(MINUTES)

Fig. 2. Denaturation of deoxyHb ( + ) , m e t H b O H ( O ) and H b O 2 control ( x ) as determined by the low-salt assay with exposure to 0 2 . Solid lines, least-squares-fitted first-order models of Table II.

significantly different from that of the fast reaction of the H b O 2 control (Table II). A second kinetic component was not observed and the final level of soluble protein was considerably higher than for denaturation of H b O 2 (Fig. 2).

Denaturation of metHbOH When the aquometHb solution is raised to p H 11.7, a ferric hemochrome is formed and a lowsalt-soluble protein appears within 30 s (Fig. 2). This intermediate is further modified to a low-salt-precipitable form at a rate not significantly different ( P = 0.5) from that of the H b O 2 control (k 1 for m e t H b O H = k 2 for H b O 2 control, Table II) and the same final level of soluble protein is approached (B 0 = B0). Thus the low-salt soluble intermediate of H b O 2 and m e t H b O H denaturation appear to be the same.

Denaturation of HbCO Provided the hemoglobin and alkaline buffer were equilibrated with 2% CO in nitrogen prior to mixing, the absorption spectrum of the reaction mixture remained that of untreated H b C O at neutral p H over the 12 h of observation. Throughout the 16 h that aliquots were tested, the protein of the reaction mixture remained soluble in the highsalt-neutralizing buffer, whether air-equilibrated or CO-equilibrated. Thus, we found no evidence of denaturation when oxygen is excluded from the reaction mixture.

90 TABLE

11

MetHbOH A N D deoxyHb D E N A T U R A T I O N

BY LOW-SALT

ASSAY

S y m b o l s and units same as in Table I. Times o f 1 m i n o r less were omitted to avoid the complication of an induction p e r i o d .

Hemoglobin derivative

Applied model

Linear parameters

Non-linear parameters ( × 103 s 1)

Fit statistics

DF

Deoxyhemoglobin

B 0 + B 1 exp( - kit)

B 0 = 0.486_+0.002

k 1 = 7.53_+0.37

S . D . = 0.00711

19

B 1 = 0.410_+0.015

(Q =1.53 min)

R 2 = 0.99244

Oxyhemoglobin" control

Bo + B I exp(-

kit)

+ B 2 exp( - k2t )

B0 = 0.2206_+0.0047

k 1 = 7.05_+0.32

S.D. = 0.00772

B 1 = 0.874_+0.029

(t 1 =1.64 min)

R 2 = 0.99811

B: = 0.1346_+0.0075

k 2 = 0.295 _+0.042

17

( t 2 = 39.2 m i n )

Methemoglobin hydroxide Oxyhemoglobin

control

B o + B I exp( - kit)

"

B 0 + B I exp( -ktt) + B 2 exp(

k2t)

B o - 0.281 _+0.010

k I = 0.240_+0.020

S.D. = 0.0232

B 1 - 0.532 _+ 0.016

( t I = 48.1 m i n )

R 2 = 0.9892

Bo = 0.2255_+0.0047

/% = 6.55 _+0.28

S.D. = 0.00846

B 1 = 0.915_+0.027

( Q = 1.76 m i n )

R 2 = 0.99837

B 2 = 0.1199_+0.0074

k 2 = 0.232_+0.042

13

17

(12 - 4 9 . 9 m i n ) " Slight differences between oxyhemoglobin controls are due to use o f different batches of alkaline buffer - rates are extremely

sensitive to slight differences in pH. The 95% confidence intervals for l o w - s a l t - a s s a y d a t a f r o m four oxyhemoglobin experiments are: k 1 = ( 6 . 4 7 _ + 0 . 8 8 ) - 1 0 - 3 s - l ; k 2 = ( 0 . 2 7 0 _ + 0 . 0 3 8 ) - 1 0 3 s 1; B0 = 0.226 + 0 . 0 0 7 8 : B 1 = 0.903 _+ 0.038; B 2 = 0.125 + 0.0094.

This is contrary to previous reports of denaturation of HbCO in alkali [9] and with heating [20]. Close reading of these papers indicates, however, that oxygen must have been present. When we added 02 to the alkaline reaction mixture, some HbCO was converted to ferric hemochrome and low-salt-precipitable protein. The spectrum reverted to the original HbCO spectrum on adding dithionite.

of the fast reaction of HbO: 20-fold (Fig. 3, Table III). A similar result was obtained by Enoki et al. [21] using hemolysates. On the other hand, attaching p-hydroxymercuribenzoate to these sulfhydryl groups increased the rate of denaturation significantly and increased the reaction order (Fig. 3, Table III).

Denaturation of mercurated HbO: and metHbOH For HbO 2, the rate of the fast reaction was unaffected by binding HgC12 to the two accessible sulfhydryls (on the F9(93)fl cysteines); however, the low-salt-soluble product was no longer detectable. The rate of the single first-order reaction was not significantly different ( P > 0.10) from the rate of the fast reaction of unmodified HbO2 (Tables III and I). For metHbOH modified the same way, the low-salt assay likewise did not detect a slow reaction, probably because the low-salt-soluble intermediate, like aquometHb, is rendered precipitable in low-salt neutral buffer by the attachment of Hg. Modifying, in addition, the 4 sulfhydryls located in the cq/3~ interface decreased the rate constant

o 80i ~) o6o

0

040

~ o~o

o oo °

<

i

1

i

,

, -

TIME (MINUTES)

Fig. 3. Denaturation of modified HbO 2 as determined by the chromophore absorbance assay. HbO 2 treated with excess HgC12 ( + ) o r excess p-hydroxymercuribenzoate ( P M B ) ( O ) are compared with an untreated control ( x ) . Solid lines, leastsquares-fitted models of Table I I I . A second-order model was used for the PMB-HbO 2, first-order models for the others.

91

TABLE Ill DENATURATION

OF OXYHEMOGLOBIN

- EFFECT OF MERCURATION

OF SULFHYDRYLS

Treatment a

Fit model

Linear parameters

Non-linear p a r a m e t e r s ( × 103 s l)

Fit statistics

DF

U n t r e a t e d (b)

B 1 exp( - kit )

B l = 1.011 _+0.014

k I = 5.78_+0.10 (t I = 2.00 m i n )

S.D. = 0.00876 R 2 = 0.99864

12

B0 + B] e x p ( - kit )

B 0 = 0.2216_+0.0027

k x = 8.08 + 0 . 4 7 (t I = 1 . 4 3 m i n )

S.D. = 0.0106 R 2 = 0.99057

17

B 1= 1 . 0 4 2 - + 0 . 0 7 4

+ 8 H g C I 2 (b)

B l exp(- kit)

B a = 0.9845 +_0.0072

k~ = 0.245 _+0.005 (t I = 47.1 m i n )

S.D. = 0.0126 R 2 = 0.99861

10

+ 12 P M B e (b, d)

B l / ( 1 + Kit )

B 1 = 0.9947-+0.0061

K 1= 38.98-+0.38 ( 1 / K 1 = 0.428 rain)

S.D. = 0.00128 R 2 = 0.9999317

17

+ 2 H g C I z (c)

a b c a

E q u i v a l e n t s of m e r c u r i a l a d d e d p e r e q u i v a l e n t of t e t r a m e r ( w h i c h c o n t a i n s six s u l f h y d r y l g r o u p s ) . T h e c h r o m o p h o r e a b s o r b a n c e a s s a y w a s used. T h e l o w - s a h w a s used. T i m e s of 1 m i n or less w e r e o m i t t e d to a v o i d the c o m p l i c a t i o n of a n i n d u c t i o n p e r i o d . S e c o n d - o r d e r m o d e l s i g n i f i c a n t l y b e t t e r t h a n f i r s t - o r d e r m o d e l ( p a r t i a l F-lest, P << 0.0001). K 1 = CoBik w h e r e C 0 is the initial c o n c e n t r a t i o n , B 1 is the linear p a r a m e t e r , a n d k is the s e c o n d - o r d e r r a t e c o n s t a n t (units, M i. s i). O t h e r s y m b o l s a n d u n i t s s a m e as in T a b l e I. c PMB, p-hydroxymercuribenzoate.

Sulfhydryl oxidation During alkaline denaturation, oxidation of sulfhydryls was observed for HbO, but not metHbOH (Fig. 4). The loss of titrable sulfhydryls proceeded with two distinct kinetic components. The first was 40% slower (significant at P < 0.05)

+ ~6o-

~o E

~_ 4o-

23 o

2 0 - - -

01

than the fast reactions we observed and accounts for two of the sulfhydryl groups (Table I, row 4: B t --2.1). The remaining sulfhydryls reacted 15fold more slowly than the slow reaction (Table I). Evidence of disulfide formation in the alkaline reaction mixture was obtained by SDS-gel electrophoresis of aliquots taken after 6 h of reaction. Bands due to globin dimers, trimers and tetramers and two types of monomer were observed with HbO 2, whereas only a single monomer band was observed with metHbOH. The latter corresponded to the slower monomer band obtained with HbO 2. All of the additional HbO 2 bands were eliminated by treatment of the samples with 2-mercaptoethanol, demonstrating that they arise by disulfide formation. This includes the faster monomer band which probably had an intrachain disulfide link. Discussion

o~5 ~!o TIME (MINUTES)

Fig. 4. S u l f h y d r y l titer of H b O 2 ( x ) a n d m e t H b O H ( + ) during denaturation. Determined by reaction of aliquots with p - h y d r o x y m e r c u r i b e n z o a t e in the p r e s e n c e of SDS. C u r v e d line, least-squares-fitted two-component first-order model of Table I. H o r i z o n t a l line, s t r a i g h t line o f s l o p e zero fitted to m e t HbOH data.

Reactions of oxyhemoglobin Our observation of two reaction steps for HbO 2 cannot be explained by differing a and fl chain stabilities [5] because the slow reaction accounts for only 13% of the denaturation. The results are best accounted for by the following linked, parallel

92 reaction sequences: f

O*

. . [I] HbO2 ~ ~ ~7 / ~ low-salt-preclpxtable o* oxidized product

~

s

[II] metHbOH ~ ferric hemochrome~ low-salt-precipitable ferric hemochrome As documented in Fig. 1 and Table I, the rapid disappearance of H b O 2 absorption and the appearance of some low-salt-precipitable protein appear to be governed by the same rate-limiting step, f. In the slow reaction, on the other hand, step s controls the rate of disappearance of a low-saltsoluble, high-salt-precipitable ferric hemochrome intermediate. The primary evidence for the above scheme is that the slow reaction of H b O 2 appears to be the same as that of m e t H b O H . In both cases a lowsalt-soluble, high-salt-precipitable intermediate is converted at the same rate to a similar low-saltprecipitable form. In both cases, the intermediate has a ferric hemochrome absorption spectrum and is rendered low-salt-precipitable by prior titration of the F9(93)fl sulfhydryls with HgC12. The slow reaction is not observed when m e t H b O H is not present or cannot be formed (e.g., during the denaturation of deoxyHb and HbCO). The formation of an oxidant during the denaturation of H b O 2 is indicated by the loss of titrable sulfhydryl groups (Fig. 4) and disulfide formation. Evidently dissolved free oxygen in the alkaline reaction mixture is not sufficiently reactive, since sulfhydryl oxidation did not occur, though oxygen was present, during 6 h of metHb O H denaturation (Fig. 4). Oxidant was formed only when both oxygen and ferroheme were present in the alkaline reaction mixture, suggesting that the oxidant is a reactive form of oxygen generated by interaction with ferroheme. The formation of superoxide and hydrogen peroxide in the autoxidation of H b O 2 has been demonstrated at neutral p H by the addition of superoxide dismutase or catalase, (not feasible at p H 11.7) [22-25]. Both superoxide and hydrogen peroxide are capable of oxidizing sulfhydryl groups of proteins [26,27] and the heme of H b O 2 [28,29]. It is thought that relaxation of steric constraints in the heme pocket during an early denaturation step

could allow superoxide to dissociate from native H b O 2 or be displaced by a nucleophile such as C I - or intrinsic imidazole [30]. The dissociation or displacement reaction would necessarily cause the disappearance of the H b O 2 spectrum; therefore, under our conditions, any such superoxide formation must occur during or in a rapid step following the rate-determining step f in the reaction scheme. The oxidation of two sulfhydryl equivalents is about 40% slower than step f, which suggests that these sulfhydryls react with superoxide nearly as quickly as it is formed. The reactive oxygen formed in sequence I must react rapidly with H b O 2 in initiating sequence II, otherwise sequence II could not compete effectively with sequence I, and the loss of HbO2 absorbance (due to sequences I and II combined) would not be observed to have the same kinetics as the initial loss of low-salt-soluble protein (due to sequence I). Both the fast and slow protein destabilization steps appear not to be caused by disulfide formation. Both components of sulfhydryl oxidation during H b O 2 denaturation followed step f and did not coincide with the slow step (Table I). The putatively identical slow step of m e t H b O H destabilization occurred in the complete absence of sulfhydryl oxidation or disulfide formation. Also, both destabilization steps occurred only when the heme was ferric or could have been ferric. During step f of H b O 2 destabilization, heme oxidation occurred at the same rate. Destabilization occurred during m e t H b O H denaturation and sequence II of H b O 2 denaturation, but not for H b C O in alkali and ferrous hemochrome (formed by deoxyHb in alkali) when 0 2 was excluded.

The rate-limiting step for sequence I The rate of alkaline denaturation of rat, rabbit and bovine deoxyhemoglobins is of the same order as that of the corresponding oxyhemoglobins, even though there are enormous differences in denaturation rate between the three species [31]. In this study we show that, for hemoglobin A, the rates of denaturation of the deoxy- and oxy-derivatives (by sequence I) are not significantly different (Table II). These observations indicate that the denaturation of both derivatives is rate-limited by an identical step which is much more sensitive to species differences than to the presence or ab-

93 sence of the oxygen ligand. Our results show further that neither autoxidation nor SH oxidation can be rate limiting in this reaction, because oxidation was impossible in the d e o x y H b reaction due to the presence of dithionite. If monomerization is rate-limiting, as predicted by Guidotti et al. [12], then the reaction should be faster after attaching the large, ionized p-hydroxymercuribenzoate group to the sulfhydryls located in the a~/3~ subunit interface. Hemoglobin so modified is k n o w n to exist as m o n o m e r s at p H > 8 [32,33]. The reaction is indeed faster (Fig. 3, Table III), and the change to second-order kinetics indicates that a different reaction step becomes ratelimiting. If ionization of the cqBi sulfhydryls in alkali increases the rate of monomerization, as proposed by Perutz, then the species differences in alkaline denaturation rate are explained [13]. Also, the lower rate we observe after titrating these groups with HgC12 (Fig. 3, Table III) is explained if the a d d u c t prevents ionization at p H 11.7. The available equilibrium data [34-36] indicate that, of the possible ligands present in the reaction mixture at p H 11.7, X in protein-S-Hg-X is greater than 99.8% OH. Furthermore, the dissociation of O H or the binding of an additional O H - is negligible at p H 11.7 [34-38]. Thus, mercurated protein SH is not charged at p H 11.7. On the other hand, sulfhydryl groups in aqueous solution have microscopic p K values of 9.1-9.5 and are 99% ionized at p H 11.7. These results with modified H b O 2 thus provide strong support for the hypothesis that the monomerization step is rate limiting in sequence I and that the rate is enhanced in alkali by the ionization of the interfacial sulfhydryls. It is likely that monomerization is also ratelimiting for deoxyHb, since the denaturation rate is the same as for H b O 2. C O - H b does not denature in alkali by any a m o u n t detectable by our assay techniques. However monomerization probably occurs, since the cq/3~ interface structure should be the same as for H b O 2.

The rate-limiting step for metHbOH and sequence 11 M e t H b O H was completely converted to ferric h e m o c h r o m e within 30 s. The first-order rate con-

stant must be at least 20-fold greater than that of H b O 2, which is inconsistent with dimer dissociation as the rate-limiting step. Inclusion of CN , which binds strongly to ferriheme and blocks ferric h e m o c h r o m e formation decreases the rate of m e t H b O H denaturation in alkali to that of HbO~ [39]. This suggests that, for m e t H b O H , sequence I becomes rate-limiting only when a faster, ferric-hem o c h r o m e - f o r m i n g step is blocked.

Acknowledgements The authors are grateful to D.L. Rabenstein for reading an earlier version of the manuscript. A.H.B. is indebted to Robley C. Williams, Jr. for introducing him 1o hemoglobin techniques during a sabbatical leave. The work was supported by a grant to A.H.B. from the National Sciences and Engineering Research Council of Canada.

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