Heme complexes of rabbit hemopexin, human hemopexin and human serum albumin: Electron spin resonance and Mőssbauer spectroscopic studies

Vol. 61, No. 1, 1974

BIOCHEMICAL

AND EIOPHYSICAL RESEARCH COMMUNICATIONS

HEME COMPLEXES OF RABBIT HEMOPEXIN,

HUMAN HEHOPEXIN AND HUMAN SERUM ALBUMIN:

ELECTRON SPIN RESONANCE AND M:SSBAUER SPECTROSCOPIC STUDIES Alan

J.

Bearden,

WI1 1 lam T. Morgan’

Donner

Laboratory, Berkeley,

Scripps

Received

Muller-Eberhard

Unlversi ty of Cal ifornia California 54720 and of Biochemistry and Research Foundation California 52037

Department Clinic La Jolla,

September

, and Ursula

20,1974 SUMMARY

The electron spin resonance (ESR) spectra of human and rabbit ferriheme-hemopexln complexes at 3O0K show an ESR absorption characterized by = 1.60, g 2.25 and g 2.86, characteristic of low-spin ferriheme9, TKe low-spin nature of the heme-iron in heme-hemopexin 1~ proteins. corroborated b the observation of nuclear hyperflne splltting in Mdssbauer spectra at 4.2 g K. The similarity of the ESR spectra of ferriheme-hemopexin wlth those of low-spin cytochromas which coordinate heme r&z two histidine residues points to a similar coordination mechanism in hemopexin. in contrast, the ESR spectra of the 1:l and 2:1 complexes of heme with human serum albumin display signals at g = 6.0 and g = 2.0, characteristic of high-spin ferrihemeproteins. Two porphyrin-binding found

in mammalian

proteins,

serum

one mole of hemeL with circulating

and reduced

resemble

those

with

recent

tion

studies via

Hemopexin,

a higher

affinity

heme to the hepatocytes

oxidized

nated

(1).

hemopexin

heme-hemopexin

of low-spin

magnetic (8)

‘To whom correspondence Research Foundat ion. ‘Abbreviations spin resonance;

circular

than

that residues. should

unlike This

the heme iron

be addressed

265

transports

spectra

of both

those

of albumin

observation

show that

at Scripps

the Clinic

IX;

(1,4),

In conjunction

in heme-hemopexin

The MCD data

binds

(2-4),

(MCD) (7) and chemical

used are: heme, Iron-protoporphyrin MCD, magnetic circular dichroism.

Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

which

albumin

Absorption

(1,6),

dichroism

have been

a B-glycoprotein

heme proteins.

indicate

two hfstidlne

(5).

and albumin,

modificais coordireduced and

ESR, electron

Vol. 61, No. 1,1974

heme-hemopexin

BIOCHEMICAL

complex

of the oxidized

known

spin

do not distinguish neither

state

the

the

residues

of methemalbumin

spin

state

involved

in

are definitely

(1,4).

are useful

heme proteins. these

but

In contrast,

nor the

Low temperature scopy

is low-spin

complex.

heme coordination

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

electron

spin

in establlshing In this

techniques

resonance the spin

communication,

(ESR) and Mgssbauer state

of the

we report

in oxidized

on the application

and human heme-hemopexin

to rabbit

iron

spectro-

as well

of

as human

methemalbumin. MATERIALS AND METHODS The apo-hemopexins purity

(6) and their

described.

of rabblt concentrations

Human heme-hemopexin

of the Behringwerke,

and human were

Marburg,

determined was also

Germany.

1:

tested

donated

RABBIT

by Dr.

G. Schwlck

was prepared

by

HEMOPEXIN

ESR spectrum of rabbit heme-hemopexin at 30°K. frequency, 9.263 spectrometer conditions were: power to cavity, 1OmW; modulation, 1OG. Protein concentration was 7.5 mg/ml.

266

for

(9) as previously

Human albumin

g = 2.86

Figure

isolated,

The GHz,

VoL61,No.

1,1974

BIOCHEMICAL

AND

BIOPHYSICAL

COMMUNICATIONS

RESEARCH

g=F6

g = 2.26

Figure

ESR spectra of human heme-hemopexin at 30’K. Trace was obtained from a sample prepared as described in Materials and Methods, and Trace B from a preparation from the Behringwerke, Marburg, Germany. The spectrometer conditions were the same as In Figure 1. The protein concentration was 13 mg/ml.

2:

zone

electrophoresis

Chemicals) (Fe

was

III)

heme

employing added

in

heme-hemopexin

with

protein

(6,ii).

and

hemeproteins

0.02

M sodium

phosphate,

with

0.05

M EDTA.

from

(a

gift

of

rabbit

7.5

of

Dr.

to

against

triple-distilled X-band

M sodium

ESR spectra

2:1

were

by

and

concentrations mg/ml.

For was

The phosphate

optical

sample

at

in was

buffer,

for

against borate,

ESR

spectroscopy, a I:1

molar

lyophilized pH 7.4,

pH 9.3,

studies 57Fe-heme

ratio after

0.05

of

spectra

4’

M sodium

employed

added

amount

absorption

0.02

&sbauer

Organic

oxidized

an equimolar

dialyzed

7.4,

(Eastman

Fully

mixing by

or

heme

ratio.

exhaustively

6.3

Caughey)

and

molar

characterized

pH

13.0

apo-hemopexin. 0.02

was

or

(lo),

obtained

Protein

W.S.

against

a 1:1 was

The

ranged

Pevikon

A

M EDTA,

85 mg

to dialysis and

then

water. were

recorded

at

267

30°K

using

a modified

JEOL

ME-IX

Vol.61,No.

1,1974

BIOCHEMICAL

spectrometer

(12)

controlled

and the

temperature

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Mgssbauer

conditions

spectra

were

obtained

(13)

under

at 4’K and 200’K.

RESULTS AND DISCUSSION The ESR spectra

taken

(Fig.

2) heme-hemopexin

2.26,

and 2.86,

iron.

These

PH 11.5

(9

-

(g = 1.75,

values

are

1.68,

2.28

heme or heme bound and rabblt

(11)

2),

fluorlde,

to denatured

protein.

state

for

of ferric b5 at

b2 at pH 12.1

human heme-hemopexln signals

due to small

at g = 4.3

amounts

The ESR spectra spectrum

by pH over

of 1.60,

cytochrome

cytochrome

the absorption

unaffected

heme-hemopexln a ligand

not display

forms

an altered

sample

30°K,

ESR spectrum

lts

treated

which

when this

the

at 20°C with

a high

was heated

to 85’~

(Fig.

for

3B) showed

to the high-spin

treatment

produced

denatured

forms

gels

shown).

Repiaclng

a ligand

(not

heme-hemopexin

wlth

complex.

Carbon

hemopexin

(1).

Missbauer and 200°K

shows only a quadrupole

form with

fluoride

monoxide,

spectra

of free

for

both

of rabblt

range

human deutero-

pH 6.3

splltting

of 2.25

(Fig.

one minute that

to 9.3

readily

3A).

However, at

This

heat

on polyacrylamide

heme iron

complexes

in native its with

ferrl-

cyanide ferroheme-

ferroheme-hemopexin

The spectrum

of Missbauer

268

heme,

and re-examined

of 6 and 2.

rabbit

mm/set

of

accessible

as is forming

4.

excess

most of the heme was

of the

in Figure

molar

wlth

of heme-hemopexln

of ” Fe-enriched

pair

complex

g values

however,

quadrupole

a five

at 30°K

is difficult

are depicted

a single

spin

ESR spectrum

converted

4.2’K

observed

and the minor

probably

were

(S = l/2)

and for

1 and 2) are

like

at g-values

The two different

(Fig.

1) and human

shown). Rabbit

dld

(14)

(15).

(Fig.

absorptions

to those

and 2.82)

heme-hemopexin

heme-hemopexin (not

similar

identical

(Figs.

rabbit

of a low spin

and 2.70)

were

and g = 2.0

show principal

characteristic

2.24

preparatlons

at 30°K of both

absorption

and an isomer

shift

taken

at at 200’K

lines of -0.20

with mn/sec

BIOCHEMICAL

Vol. 61, No. 1,1974

Figure 3:

relative

Effect of fluoride on the ESR spectra of rabbit hemehemopex 1n . The ESR spectrometer conditions were the same as in Figure I. Trace A was obtained at 30°K after exposure of the protein to a five molar excess and Trace B of sodium fluoride at room tern erature after heating the sample to 85 g C for one minute and re-examinlng it at 30°K.

to a 57 Fe:Pt

values

of quadrupole

source

for

low-spin

spectrum

does not allow

state.

features position;

this

in other

low-spin

(Fig.

prominent

g-values

compounds

(13,16);

a definite

assignment

at g, = g

5B) Y

the

nevertheless, spin

pattern

These

range thls

or oxida-

shows additional at the 57Fe

hyperflne

effect

shown

(16).

the ESR spectra (Fig.

are withln

Interaction

to the nuclear

ferrihemeproteins

to Fe metal.

of either

absorption

hyperfine

similar

referred

shift

of a nuclear

5A) and 2:l

ne~‘s+z

and isomer

the MbJssbauer

is quite

In contrast, a 1 :I

Fe (III)

At 4.2’K, indicative

or +0.27

splitting

expected

tion

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of human serum albumin ratio

of heme to protein

= 6 and g, = 2.

269

at 30°K with

The observed

show ESR signals,

Vol. 61, No. 1, 1974

BIOCHEMICAL

e

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mm/set

9=2

g=6

1

-1

270

VoL61,No.

which

were

typical any

BIOCHEMICAL

1,1974

proportional

of

high

rhombic

seen

spin

of

12’K

and

conclusion,

reported

here

the

is ESR

known

is independent

have

dihistidyl

that

the

evidence

amount

of

ferric

hemeproteins

heme

No

heme.

ESR

and

the

RESEARCH

bound

low-spin

Mgssbauer

to

the

and

do

Fe

nature

(7),

low-spin oxidation

(III)

heme in

of of

the

state.

ferriheme-hemopexln

heme

spectra

low-spin

also of

of

BIOPHYSICAL

COMMUNICATIONS

protein, not

are

indicate

signals

were

lOOoK.

demonstrate

spectra

to

the

the

ferroheme-hemopexin protein

the

(S = 5/2)

distortion

between In

to

AND

ferriheme-hemopexin

this

spin

state

Further, to

those

(17),

coordination

heme-hemopexin

is

of

the of

Since

complex. this

resemblance

cytochrome provides

similarly

hemeof

b5

(lb),

additional coordinated.

ACKNOWLEDGEMENT This Foundation Laboratory,

research was supported by grants from the National Science (GB-13585), the Atomic Energy Commission through the Donner and the National Institutes of Health (HE-08660, HO-04445, AM-16737). A.J.B. is the recipient of a Career Development Award #GM-24,494, U.M.E. of Career Development Award #AM-16,823, and W.T.M. of post-doctoral fellowship #AM-53050 from the National Institutes of Health. 57 Fe-heme We are indebted to Dr. Winslow S. Caughey for the gift of and to Susan Wormsley for expert technical assistance.

REFERENCES

1. 2.

Mu1 ler-Eberhard, (in press). Seery, V.L.,

U., and

and

W.T.

Morgan,

Muller-Eberhard,

U.

(1374) (1373)

Ann. J.

N.Y.

8141.

Acad.

Sci.

Chem.

248,

J.

Biochem.

3796-3800. 3.

Hrkal,

2,

Z.,

Vodrgka,

Z.,

and

Kalousek,

I.

(1974)

Eur.

73-78.

Figure

4:

&ssbauer spectra at 200°K

Figure

5:

ESR are: (B) the was

spectra of we-t-e obtained (upper trace)

57 Fe-heme-hemopexin. rabbit on 85 mg of lyophllized and at 4.2’K (lower

The material trace).

spectra of human methemalbumin at 30’K. Shown (A) 1 :l molar ratio of heme to protein and 2:l ratio. ESR spectrometer conditions were same as in Figure 1. The protein concentration 8 mg/ml.

271

Vol. 61, No. 1, 1974

4. 5. 6.

I: 9. 10. 11. 12. 13. 14. 15. 16. 17.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Beaven, G.H., Chen, S.-H., D’Albis, A., and Gratter, W.B. (1574) Eur. J. Biochem. 2, 535-546. Huller-Eberhard, U., Bosman, C., and Liem, H.H. (1970) J. Lab. Clin. Med. & 426-431. Hrkal, z., and Mulier-Eberhard, U. (1971) Biochemistry 10, 1746-1750. Vickery, L.E., and Morgan, W.T., manuscript In preparat‘f;jn. Seery, V.L., Morgan, W.T., and Muller-Eberhard, U., manuscript in oreoaration. seery, V.L., Hathaway, G., and Muller-Eberhard, U. (1372) Arch. Biochem. Biophys. l& 269-272. MBller-Eberhard, H.J. (1360) Stand. J. Clin. Lab. Invest. l2, 33-37. Morgan, W.T., and Muller-Eberhard, U. (1572) J. Blol. Chem. 247, 7181-7187. Bearden, A.J., and Malkin, R. (1972) Biochim. Blophys. Acta 3, 456-468. Moss, T.H., Bearden, A.J., Bartsch, R.G., Cusanovich, M.A., and San Pietro, A. (1368) Biochemistr 7 i53i-1586. Bois-Pol toratsky, R., a73+ an E ren erg, A. (1967) Eur. J. Biochem. 2, 361-365. Watari, H., Groudinsky, O., and Labeyrie, F. (1867) Biochim. Biophys. Acta 131, 592-594. -Caughey, W.S., Fujimoto, W.Y., Bearden, A.J., and Moss, T.H. (1966) and Argos,

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