Nonenzymic protoheme formation

Vol. 25, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH CO/vVvKJNlCATlONS

NONEXZYKICPROTOHEW FORMATION Rikio Tokunaga and Seiyo San0 Department of Public Health, Faculty of Medicine, Kyoto University, Kyoto, Japan

ReceivedOctober 6, 1966 The final protoporphyrin

i.e.

step in hemebiosynthesis, ring,

the chelation of iron into

seemsto be catalyzed by the enzyme in liver

mitochondria and in the particles

from hemolyzed chicken or duck erythro-

cytee (Labbe, Hubbard end Caughey (1963); Tamai, Yasucla and Yoshikawa (1965)).

Porra and Jones (1963)i

These authors, however, demonstrated

that the nonenzymic protoheme formation from protoporphyrin compared to the oase of the enzymic reaction. Lockwood and Rimington (1958) enzymically

Yoneyama,

was negligible

On the other hand, Eeikel,

reported that protoheme was not formed non-

from protoporphyrin

and ferrous iron but when native globin or

teepol was added in order to solubilize

the porphyrin there was an appreci-

able formation of protoheme. Nevertheless, they reported that a high degree of reproducibility

was not possible.

Granick and Mauzerall (1956)

also reported that protoporphyrin

and ferrous iron would readily

combine

without enzyme8 under appropriate

conditions but no supporting data sas

presented. In this communication we wish to present a definite

evidence of highly

reproducible nonenzymic protoheme formation frcxa protoporphyrin

and ferrous

iron in the presence of sodium dithionite

under physiological

condition end

also describe the presence of sameactive

ferrous iron compoundaontaining

aulfur which might be considered to plsy an important role on the ahelation of

iron into protoporphyrin

ring.

489

Vol. 25, No. 5, 1966

BIOCHEMICAL

EXPER~~ALkETHOD

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The aqueous aolution of protoporphyrin

with ferrous iron at 38' C under strictly

anaerobic conditions and the

protoheme formed was estimated as pyridine

hemochromogen. Protoporpbyrin

(2 mg) ww dissolved in 5 ml of 1 M Tris-FICl buffer, temperature, then the solution was centrifuged to remove the insoluble protoporphyrin. solution and diluted

photometrically

at 15,000 r.p.m.

Protoporphyrin

3 ml of 1 M Tris-HCl buffer,

way.

in Thunberg tubs with

Protoporphyrin

(0.15 pole)

in

pH 8.2, was placed in the main arm of Thunberg

and ferrous smmonium sulfate

(7.50 poles)

The stock solution

solution with a different

and pH values was prepared in a,similar

mechenical shaking at 38' C in the dark.

with the

was determined speotro-

at 408 7 in 1 N HCL (d 0.1 pr)d- 24.0).

The incubation was csrried out anaerobically

tube,

for 20 min

The supernatant was used as a steak

of protoporphyrin

should be used within three days. concentration

pH 8.2, at room

before the use to the desired ooncentration

The concentration

mme buffer.

was incubated

(5.25 polea)

and eodium dithionite

dissolved in 1 ml of the asmebuffer was placed in the side

BRA. Oxygen was removed by two or three cycles of freezing under vacuum (< 0.02 mmHg) with an intermittent

and thawing

flushing with nitrogen.

Then the contents of both anus were mixed and incubated by the following two different

methods; lethod-1,

C and incubated; Method-P, the

the contents in both sumswere mixed at 38' tube

was incubated at 38' C for 60 to 90 min,

then mixed, followed by inoubation. After

the incubation,

the reaction

of the mixture of ethyl acetate-glacial mixture was transferred Protoporphyrin until

was stopped by the addition acetic acid (Jtl,

v/v)

and the

to a aepsratory funnel, washed with distilled

was extracted

of 40 ml

water.

from ethyl acetate with each 5 ml of 2.5 H EC1

no fluorescence remained in ethyl acetate leyer.

acetate layer containing protoheme was washeddth evaporated to dryness under reduced pressure.

490

The remaining ethyl

distilled

rater and then

The protoheme wae diesolved

BIOCHEMICAL

Vol. 25, No. 5, 1966

in pyridine-0.1

H NaOHsolution

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(20r80, v/v)

and estimated as pyridine

hemochrcnnogen. RESULTS snd DISCUSSION --

It wss clearly

demonstrated that protoheme a&8

obtained nonenzymioally by the mere incubation of protoporphyrin ammoniumsulfate

in the presence of sodium dithionite.

and ferrous

The formation of

protoheme increased uith time end reached to maximumafter

approximately 30

to 40 minutes (Fig.

1).

Maximumformation of protoheme was constantly

obtained in a yield

of 30 to 35 $ over the piI range 7.8 - 9.6 with a high

degee of reproducibility HCl buffer

under the ssmeconditions (Fig.

1 and 2).

(1 Y, pH 8.2) uas found to give the highest yield

but Tris-HC1 buffer (O.lM, or barbital-HCl

buffer

fl 8.2) ,wleate-I4aOH

buffer

Tris-

of protoheme,

(0.05 I,

pH 8.2)

(0.05 M, DH 8.2) yielded 20 $, 7 $ and 3 $ of proto-

hemein one hour, respectively.

IO

incubation

time

Imln)

20

Incubation

30

40

30

timr

00

I20

lmln)

Fig. 1. Non-ensymic Protoheme Formation frau Protoporphyrin. A: Prot.ohemeformation from low concentration of protoporphyrin. 0,15 pole protoporpbyrin, 5.25 poles ferrous iron, 7.5 poles Na&O4. B: Protoheme fowstion from hi@ concentration of protoporphyrin. 1.0 urnmole protoporphyrin, 35 poles ferrous ircm, 50 poles Na25204. Both A and B were incubated anaerobically in 4 ml of 118 Trie-HCl buffer, pH 8.2. a,b nfth preinoubation of ferrous iron and Na2S204(ldethcd-2). a’,b’ ----a without preinoubation of ferrous iron and Na2S204(Method-l). a",b" -a-*- without Na2S@4 in Method-l.

491

Vol. 25, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Protoporphyrin

added

(mu moles)

2. At Protoheme formation at 60 minutes incubation versus pH. The low concentration of protoporphyrin was used. Br Protoheme formation at pH 8.2 from various concentrations of protoporphyrin at 60 minutes incubation. with preincubstion (Method-P), ----- without preincutition (lethoci-1). Fig.

Three important evidences were found in nonenvic 1)

The presence of sodium dithionite

protoheme was obtained in the absence reducing agents such aa cysteine, sulfate,

sodium metabisulfite

sodium dithionite.

2)

dark

of sodium dithionite.

However, other

ascorbic acid, glutathione,

sodium thio-

snd sodium borohydride could not replace

Protopor*in

is not acting as

was not reduced by sodium

in this experiment.

A prominent formation of black precipitate

incubation.

In the absence of sodium dithionite,

always took place during the neither

black precipitate

nor protoheme was observed (l?Lg. 1, A and B, curves atI and b"). clearly

No

is essential in the formation of some complex with

iron which is described below. in the

for the reaotion.

It appears thus that sodium dithionite

the reducing agent but it

dithionite

was essential

protoheme formation.

As

shown in Fig. 1, A and B, protoheme formation in Method-l (curves

a' and b*) appeared with a lag phase (20 to 30 min). precipitate

After

that time, black

appeared with the formation of protoheme. However, on the

492

BIOCHEMICAL

Vol. 25, No. 5, 1966

preincubation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

experiment according to Method-2, the black precipitate

formed from the incubation of ferrous iron and aodium dithionite of Thunberg tube end, after

the miring,

was

in one B;IIp

protoheme formation occurred

promptly without the lag phase as shown in curves a and b. These observations indicate

that the condition

was formed seemedto be absolutely black precipitate

essential for protoheme formation.

The black precipitate

(presumably labile

sulfur)

black precipitate,

H2S was liberated.

finely

3)

or

wss found to contain sulfur

and ferrous iron.

On addition

Sodium borohydride,

of acid on the however, madea

but this was not incorporated into protoporphyrin.

ground ferrous sulfide

sodium dithionite

NO

was formed from ferrous iron and sodium thiosulfate

sodium metabisulfite.

black precipitate

when black precipitate

The

or reduced iron powder in the presence of

reacted with protoporphyrin

to give protoheme.

It is an important evidence for heme formation that protoporphyrin

involved by aggregation in euch a reactive sulfur compoundo The failure

is

environment containing iron-

of protoheme formation by the addition of

detergents such as Tween 20 or l&as01 4130 could well be due to eolubiliaation removing the porphyrin from the environment mentioned above. findings seem to oppose the hypothesis that the solubilimation porphyrin is essential for iron inoorporation. sodium dodecyl sulfate, pyridine

protoporphyrin

protoporphyrin

and Z,+diacetyl

converted to their

was oonverted to the hemeof which

deuteroporphyrin

behaved similarly

to

into protoporphyrin

mesoporphyrin and porphyrin c were more

corresponding hemesthan protoporphyrin

the absence of aodium dithionite.

aad

However, in the presence of

as regards the absolute requirement of sodium dithionite.

However, copro-, hemato-, deutero-,

ration

of proto-

hemochromogen had an absorption maximumat 552 mu in place of 557 mp.

2&diformyl-

readily

These

Therefore,

may be different

this may be due to the electrophilic

positions 2 and 4 of the porphyrin ring.

493

even in

the mechanismof iron incorpofrom that of other porphyrins

character of double bonds in The amounts of protoheme formed

Vol. 25, No. 5, 1966

from protoporphyrin

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in nmenaymio eyetem were found to be in good agreement

with those reported in enzymic system (Labbe, Hubbard end Caughey (1963); Porra and Jones (1963); Yoneyema, Temai, Yaeuda end Yoehikawa (1965)). Further inveetigation

on ferroue sulfur

compoundfound in We

eeemeto be more important than the discussion

experiment

whether iron incorporation

is nonenzymio or enaymic.

Lebbe, R. F., Hubbard, B. end Cau&ey, I. Porra,

R.

S., Biochemistry,

J. end Jones, 0. T. G., Blochem. J., a,

Yoneyams, Y., Tan&, A.,Yamda,

2, 372 (1963)

186 (1963)

T. andYoehikawa,H.,

Blochim. Riop@j.

Acts, 105, 100 (1965) Heilcel, T., Lockwood, W. H. end Rimington, C., Hature, Granick, S. and Mawsetil,

D., J. Riol. Chem., &

494

182,

313 (1958)

1119 (1958)