An electron paramagnetic resonance study of nitric oxide hemoglobin derivatives

An electron paramagnetic resonance study of nitric oxide hemoglobin derivatives

COMMUNICATIONS hot~rs in 3 ml of 0.0-1 M aspartic acid in 0.1 JI phosphatc bnfier, pH 7.4. Activity blanks were used for eacnh incubation. Lrvels of A...

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COMMUNICATIONS hot~rs in 3 ml of 0.0-1 M aspartic acid in 0.1 JI phosphatc bnfier, pH 7.4. Activity blanks were used for eacnh incubation. Lrvels of AAT were determined by measuring ihe oridattion of NAl)H by the malate dehgdrogenase at 310 mp in :I Gary II type spectrophotometer. For the kinetic measlwements the bands contail)ing 1 he active fractions were transferred to :I conical 11y1on net fixed on :I plastic handle al~tl il~trotll~wtl illto the reaction mistrlres. A 15srco~ld illcllbation was performed, and thereafter the l~aud was rcmovcd for 15 seconds io allow reading. 111 addition to the media used for preincltbai ion, olw reaction nlixture consisted of 0.05 ml of 0.012 >f NADH, 0.05 ml of malnte detrydrogenase, 0.25 mg/ml, and 0.10 ml of 0.25 31 2~osoglllt:tr:ttr~. The reaction was started by the addition of I he 2.oxogl~~tnrnte. The method of 1,ow-y et (11. (IO) LV:IS lised for protein determiwlitim. The spccitic activities are expressed in millimolts of ?J9l)H osided (or SAl) formed) br the malate dehydrogen:tsc per milliliter per mimltc per milligram of protein for ilAT isozymes, and the rat.io between their activities is shown in Figs. l-3. The pat t ems of these isozymes are different in each of Ihr organs. In heart (Fig. 1) both isozymcs inrrease in the same proportion, and i he ratio t hrollghout, the postnatal development is :tpproximately constant. In the kidney (Fig. 2) the two isozymes show a very slight and parallel increase of specific activities starting on the twelfth day; thlls their ratio throllghout, the postnatal development also remains constant,. Tn brain (Fig. 3), on the contrary, the specific activities are characterized by a lo-fold increase from birth t.o adulthood for the extrnmii ochondrial enzyme aud a j-fold increase for the mitochondrial enzyme; however, the latter act,ivity remains extremely low. Similar ratios were found when DN4 (11) was used as reference for brain and kidney. Otlr results show that, t,he maturation of the AAT isozymes follows different. rhythms in the three organs. The interpretation of the specific* metabolic role of the est.ramitochondrial and mit,ochondrial enzymes requires further investigation. REFERENCES 1. BORST,

P.,

AND

PEETERE,

E.

&I.,

Hiochim.

Biophys. ;Icla 54, 188 (1961). 2. HOOK, R. H.. AKU TESTLING, C. S., Biochim. Biophys. Acta 66, 358 (1962). 3. ;\~ORINO, Y., ITOII, H., .4iXD WAD.4, HI., BiOthem. Biophys. Hes. Commun. 13, 348 (1963). 1. NISSELBAUM. J. S., AND BODAXSK~-, O., Federation Proc. 22, 241 (1063).

203 5. Bon), G. Bow),

,J. W., Hiochem. J. 81, 334 (1961). J. W., Biorhem. J. 81, 39p (1QGl). 7. NISSELB.+LTX, J. H., ASD BODAXSKY. O., Biol. C’hem. 239, 1232 (1964). 8. HEMOX, chcmistr,q

c.

9. 8cwa~~z, BODANSKY,

I'., AND CLEL.~ND, 3, 338 (lQ64). 1\f. O.,

\T'.

Iv.,

J.

Ti., NISSEI,B.4UM, -1~. .I. Clin.

nio-

s.,

Pathol.

J.

.\?in

40,

103

(lQG3). lo.

T,OKRY,

(1.

H.,

ROYEBROUGII,

s.

,I.,

P.4RR.

A. I,., AXE ~:AsI).\I,I,, I<. J., .T. f
Received

.lzlgtrst

8, 1967; accepted

October

I:ESI)OS1

27,

1.967

-

1 Fellow vestigacion

An

Electron Nitric

of the Institllto National Cientificn, LIesico.

Paramagnetic Oxide

Hemoglobin

Resonance

de la

In-

Study

of

Derivatives

Since t,he method of isolating 01 (Hh 01) and 0 (Hb 0) subunits from human adult hemoglobin (Hb A) was established (1, 2), differences in physicochemical properties of the subunits and betu-een the subunits aud their native or reconstitut,ed parent molecule have been demonstrated (2-5). On the other hand, studies on nitric oxide (NO) hemoglobins by previorls investigators (6-Q) disclosed that an odd electron localizes on the nitrogen atom of PU’O and results in an electron paramagnetic resonance (EPR) spectrum. The shape of the spectrum depends on the primary struct.ure of the globin moiety of the hemoglobin (7). It seems very promising, therefore, to study an EPR spectrllm of Hb A and its constituent subunits for the elucidation of the nature of their functional diflerences in terms of magnetic properties. Adult hemoglobin was prepared from the blood of normal human adlIlts. Hemoglobin 01 and p were obtained according to the method of Tyuma et ul. (2) and freed from p-hydroxymercuric benzoate (p&113) completely. Their NO derivatives in a concentration of 1-3y0 in 0.1 M phosphate buffer (pH 7.4) were examined by a \‘arian V-4500 EPR spectrometer. The measllrements were carried out either at room temperatlwes or a.t low tempera-

204

COMMUNICATIONS

resonance specconditions as

similar t,ype of spectrum was also observed in NO derivatives of y chains isolated from human fetal hemoglobin. In contrast, the spectrum of NO-Hb 01 shows a marked anisotropy in g- and A-value. An addition of the spectra for the 01 and p subunits yielded a spectrum which is almost, identical with that for Hb A. Combination with pMB strongly modified the spectrum of NO-Hbol (Fig. 2), but it did not affect the line shape of NO-Hb @ significantly. The EPR absorption decreased with t,he increase of temperature without changing it,s line shapes. In all the Hb’s a plot of log S (doubly integrated area) vs. temperature (10) falls on a same straight, line (between -160 and --7O”), the slope of which coincides with that, for a frozen solution of CuSOa. Electron paramagnetic resonance signals of all the NO-Hb’s are also detectable at room temperatures, although the line shape difference among them becomes less distinct. The saturation behavior against applied micro wave power is similar for all the NO-Hb’s; EPR signals are nonsaturable at bot,h low and room temperatures. These results indicate that the relaxation mechanism and temperature effect are the same in all the NO-Hb’s. Thus, the line shape differences between 01 and p chains observed can be related to the alteredorbital configurationof heme-NO part in the subunits. For example, according to the “parallel to heme” t,heory proposed by Griffith (11), a subtle change in heme-NO linkage in subunits of different structures will induce a marked alteration in magnet,ic susceptibility and EPR spectrum of the NO-derivatives. A computer analysis t,o evaluate exact g- and ri-value of the EPR spectra is under progress. The authors are indebted to Dr. N. Maeda for giving them isolated y chains and to Mr. M. Chikada for his skilled t,echnical assistance. The research was supported in part by a Grant-in-Aid for Fundamental Scientific Research from the Ministry of Education.

tures with an anerobic tube and a variable temperature apparatus. Figure 1 shows the low temperature (-160’) EPR spectra of NO-Hb A, -Hb CY, and -Hb p. Clearly the line shapes and apparent g-values of the spectra are different from each other. The spectrum of NO-Hb A is essentially similar to the reported line shape (6, 7) and to the spectrum for reconstituted Hb A (01 + p). The spectrum of NO-Hb p is nearly symmetrical, and its g-values are probably very close. The anisotropic hyperfine coupling constant, A,, estimated from the second derivative curve, was about 17 gauss. A

E., AND FRONTICELLI, C., J. Biol. Chem. 240, PC551 (1965). 2. TYUMA, I., BENESCH, R. E., AND BENESCII, R., Biochemistry 5, 2957 (1966). 3. ANTONINI, E., BUCCI, E., FRONTICELLI, C., WYMAN, J., AND ROSSI-FANELLI, A., J. MoZ.Biol. 12, 375 (1965). 4. BEYCHOK, S., TYUMA, I., BENESCH, R. E., AND BENESCH, R., J. Biol. Chem. 242, 2460 (1967). 5. OHNISHI, S., MAEDA, T., ITO, T., HWANG K-J., AND TYUM~, I., Unpublished experiments.

EPR

spectra

of

Hb’s

FIG. 1. Electron paramagnetic resonance spectra of NO-Hb LY, -Hb 0, and -Hb A. Experimental conditions: 0.1 M phosphate buffer, pH 7.4; -160”. Span of arrow corresponds to 33.2 gauss; vertical line shows the position of DPPH signal.

FIG. 2. Electron paramagnetic 7 tra of ?rO-Hb (Y~~IB. Experimental in Fig. 1.

REFERENCES 1. BUCCI,

COMPIIUNICATIONS J. E., Dis19, 140 (1955). 7. GORD~. W., .&SD REXFORD, H. pi., in “Free Radicals in Biological Systems” (RI. 8. Blnis Jr., H. W. Brown, R. M. Lemmon, Ii. 0. J,indblom, and 31. Weissbluth, rds.), p. 203. Academic Press. New York (1961).

8.

INGRAM,

cussiona

8. S;AXVIER.

D.

J.

E.

AXD

Faraday

K.

FREEMAN,

G.,

AND

TAKESHI

;\IILLS,

Fumarate

AND

-

for

70”

-0Od

‘H

F--F-oH “-F-7

5.0

, mw

=

443.

5 min 10 min 20 min

41 GG7

(Xi .5 41.; 21.2

65.1 40.0 21.0

optical density was observed instead of a decrease as with fumnrate. This allowed the development of an assay for the enzyme in t,he presence of fluorofumarate as substrate. Addition of 2,4dinitrophenylhydrazine solution to the incllbatiou mixture gave a precipitate of a derivative, a react,ion not observed with the control samples which contained no enzyme. Likewise, when enzyme denatured by prolonged heating was incubated with fluorofumarate, no reaction occurred. When the precipitated derivative was chromatographed on a thin layer of silica gel (Eastman Chromagram sheet) in a solvent system previously developed (7), the major product had the same KF as oxaloacetate-2,&dinitrophenylhydrazone together with a minor component with the same HP as pyruvate-2,4-dinitrophenylhydrazone. The pyruvate apparently arose from the spontaneous decarboxylation of osaloacetate. Hence, the reaction catalyzed by flunarate hydratase lmder these circumstances seemed 1.0 be the following (Eq. 1):

yo-

F ,C,co~ HOH-

Fluorofumarate

n Measured in 0.033 M sodium phosphate buffer, 7.3, and 25’. b y0 activity remaining after enzyme was heated at 50’ for vnriolls time periods.

Flmnrate hydratase (EC 4.2.1.2) has been studied extensively and found to act only on the pair of slll)strates, L-malate in one direction and fumarnte in the other (1, 2). The fluoro group is similar in size to the hydrogen atom, and this has led to the successful development of metabolic antagonists in which fluorine is substituted for hydrogen (3a, b, 4). The study of flnoro-analogs of citric acid cycle intermediates has shown that most of t,hese are far less tosic in animals t.han is fluoroacetate. This led us to examine the action of fumarate hydrat,ase from pig heart cm flllorofnmarate, and we report here that this compound is a substrate for the enzyme. Fluorofumaric acid was prepared from 2,2-di-

+

Substrate Fumaraie

pH

Hydratase

II c

OS

FLUOROFUX~RLTE

Data

y0 Sctivityb

THIGH

I

OF FUM.ZR:~TE II~DR.~T.~sE ACTIVITI. HEITISG ASS.IyED WITH FCMhR4TE

V,,, , rmoles/ml/rniIl/rllg

KUN-JOO Hw-~NG ITIR~ TYUM.Z

Substrate

Loss

K,,,

J. S., Science 137, 752 (19ti2). 9. REIN, H., RISTAU, O., AND JUXG, F., Polia Hacrnalol. 82, 191 (1964). 10. ~:HRE?I;REXG, .A., z~~rkiv k’ez!li 19, 119 (1962). 11. GRIFFI-121 , J. P., l’roc. ROJ. Sot. (Londoll), Ser. .I 235, 23 (1956).

Fluorofumarate-A

TABLE

BENNETT,

sot.

?\I.,

20.5

-I

c=o CH,

coo

fluorosuccinic acid by the method of Raasch et al. (5). When fluorof\lmarat,e was incubated wit.h a crystalline prppnrat,ion of fumarat.e hydratase from pig heart (Boehringer Mannheim Corp.) rmder the assay conditions described by Racker for fumarate (ci), and the reaction followed in a spectrophotometer at 300 ml*, a rapid increase in

I

H+

+

F-

(Eq

1)

koo-

To demonstrate that this reaction was not catalyzed by some other enzymic activity, which might have been present as an impurity in the enzyme preparation used, partial heat inactivation of the enzyme preparation was studied, and the inactivated preparations were separately incubated in the presence of fumarate or fluoroflunnrate. The results are presented in Table I.