Reaction of polymer-heme complexes with carbon monoxide or molecular oxygen

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REACTION OF POLYMER-HEME COMPLEXES WITH CARBON MONOXIDE OR MOLECULAR OXYGEN Eishun

Tsuchida,

Etsuo

Hasegawa,

Department of Polymer Waseda University,Tokyo

Received

July

Summary molecular

oxygen

were

monoxide

and its

studied

Hill's

on the

From the

In the reaction

with

polymer-ligands

except

the reaction

of the additive

of the

with

with

poly-L-lysine

oxygen,

poly-L-lysine

effects,

it

nay play

monoxide

It was found

studied.

was determined

molecular

polymer-ligand

carbon

or

and manometrically.

also

of the complex

for

results

conformation

curve

were

parameter

plots.

complexes

spectrophotometrically reactions

equilibrium

cooperative

However,other

Chemistry, 160,Japan

of some polymer-heme

of additives

carbon

Honda

25,1975

Interactions

Effects

and Kenji

it

the

was sigmoid

to be 2.1 by the

was also

showed

that

sigmoid.

no cooperativity.

was suggested

that

an important

part

reported

formations

a helical in raising

cooperativity. Introduction

The present

of heme with tion

and in

ture

of the Method pyridine, were

In this with

conformation

Structures photometrically

we report

and formation using

oxygen

that

polyethyleneimine

is,

and N,N-dimethylformamide constants

the method

at room tempera-

and discuss

polymer-heme on the

roles

found.

poly-L-lysine,

poly-4-vinyl-

of branching Hemin

were

and Dorough

= 0.42

)

( ferri-proto-

of Willstaetter

of heme complexes of Miller

forma-

a reversible

of these

(1,3).

by the method

complex

solvents

( degree

papers

of complexes

and found

in a cooperativity

) was prepared

(pH12.0)

(1,2,3)

interactions

or molecular

to the previous

IX chloride

in the

in organic

Polymer-ligands,

according

buffer

oxygen

complexes

of polymer-ligand

and branched-type

porphyrine

characteristics

molecular

monoxide

and Materials

prepared

Phosphate

with

communication,

carbon

recently

and their

of the polymer-heme

(1).

complexes

polymer-ligands the reaction

oxygenation

authors

(4).

0.05M

used as solvents.

were (5).

determined Absorption

spectroof

Vol.67,No.3,1975

carbon

BIOCHEMICAL

monoxide

manometer

or molecular

reaction

and Discussion

between

phosphate

after

axial

one with

of carbon

monoxide

complex

hyperbolic.

with

poly-L-lysine coefficient

of the poly-L-lysine line

l(B)

log(Y/(l-Y)) a cooperative

interaction

poly-L-lysine. observed

= log

As shown in the

between in Figure

absorption

plots

(6).

K + n log

PC0

heme complexes

of molecular

1

and Hill's unit-mole/l, molecular

Carbon plots

monoxide

(B) in phosphate

[hemelo= nitrogen

equilibrium

3.0 mmole/l,

was kept

constant

complex as

from

This

behavior

absorption

but for

large

curve

others

were

a reaction,n)

the slope

situated

value

of the indicated

on an a-helical reaction

was also

by the poly-L-lysine-heme

Pco(mmHg)

Figure

the

the

a cooperative

oxygen

optically

monoxide

parameter

to be 2.1

2, such

that

Only

of heme

heme in the absence

to be sigmoid,

(a cooperative

by Hill's

region

1 shows an absorption

complex.

system was determined

in Figure

while

on the

in a 0.05M

still

and carbon

Figure

was found

were

indicated

of poly-L-lysine of heme.

monoxide

bands

results

measured

in a Q-band

monoxide,

by a poly-L-lysine-heme

The flill's

straight

iron

were

and carbon observed

carbon

These

group

of a central

changes

with

active.

c-amino

spectra

and the absorption

reaction

was not

ligands

by the

by the use of a Warburg's

absorption complex

Spectral points

the

of poly-L-lysine is

Visible

(pH12.0).

isosbestic

even

formed

was determined

a poly-L-lysine-heme

buffer with

active

oxygen

RESEARCH COMMUNICATIONS

and by a spectrophotometer.

Results

occured

AND BIOPHYSICAL

complex,

log PC0

curve buffer Total

of poly-L-lysine-heme

(pH12.0) pressure

(760 mmHg).

865

at 25'C. of carbon

complex

[polylysine]o= monoxide

(A) 35.5

and

Vol. 67, No. 3, 1975

since

BIOCHEMICAL

n was equal

only

to 2.0.

in an equilibrium Subsequently,

were

studied.

This

revealed

reaction effects

but

of types

When random-coiled

and branched

were

since

behavior

dependent

on polymer-ligands

original

structure

of each complexes

axial

a cooperative

ligands

Therefore,

from

interaction

: (a)

a regular

of a heme complex

the fact

that

with

an a-helical

increased

after

the

partially

destroyed

chain

(3),

is,

it

1).

were

number

formation

in others

with

in the

in has two in Table

because

reaction.

in its

a

cooperative

and (b)

2 before

a struc-

Actually,

heme complex

although

it

had been

a heme due to steric

hinderance

8

Figure

2

Absorption

and the effect unit-rnnole/l, Total

pressure

of molecular

of additive [hemelo= of oxygen

oxygen

by poly-L-lysine-heme

polyethyleneglycole

6.0 mmole/l, and nitrogen

(0).

866

constant

complex

[polylysine]o=

[polyethyleneglycole]o= was kept

1.

former.

to cause

coordination

monoxide,

in

summarized

than

of a polymer-ligand

by the complex

Such a different

complex

may be important

carbon

was

of polymer-ligands

conformation

with

poly-4-vinylpyridine reaction

was retarded

of poly-L-lysine

conformations

by the differences

which system

oxygen

that

content

reaction

that

a poly-L-lysine-heme

by molecular

1 suggest

and their

and conformation

only

not

kinetics.

no cooperative , was small (Table

in a poly-L-lysine

reaction in Table

in an absorption

may be caused

the same polymer

The results

ture

reported,

was .rdised

used,

parameter

P5g was higher

ligand-exchange

also

a cooperativity

polymer-ligands,

polyethyleneimine,

As previously

that

RESEARCH COMMUNICATIONS

of polymer-ligands

observed

solution.

AND BIOPHYSICAL

(e)

45.5

60 unit-mmolell, (760

mmHg).

BIOCHEMICAL

Vol. 67, No. 3, 1975

between

heme and cylindrical

or reduction monoxide coordination chain

lysine-heme

complex

measurement

of solution

after

systems

act

aggregates,

were

in Table

Therefore,

but are

added 2.

able

cases,

was made to be instabilized no spectral

changes

Table

1

molecular Polymer-ligand

in

carbon complex

with

by polymer-heme

or

was

absorption

behavior polymers, to form decreased

an cl-helical

of polymer region

aggregates and after

absorption

of

(7). the

which polymer as

And addition

at 30°C

Solvent

Amax

n

poly-4-vinylpyridine

418

1.0

1 . 1x103M-1

DMF/MeOH

1.1

58

poly-L-lysine

431

2.0

2 . 5x108M-'

pH12 aq.

2.1

130

branched

424

1.0

7 . 5x104M-1

pH12 aq.

1.0

100

polyethyleneimine

of

conformation

before

complexes

in the

conformation.

poly-L-lysine

that

K(20'C)

by viscosity

parameter

on the

1 are formed,

in the poly-L-

When various

a cooperative

visible

number

may be caused

on the

was found

or branched

of polymer-ligand

structure

n

P50(mmHg)

n : Cooperative parameter ; n : Axial coordination number of heme complex K : Formation constant of heme complex ; DMF :N,N-dimethylformamide ; MeOH : methylalcohol ; DMF/MeOH=7/3(v/v) ; Xmax Absorption maximum of deoxy-heme complex

867

with

the polymer

was confirmed

changes

to interact

of polymer-ligands

oxygen

through

complex.

by the formation

was observed

Effects

it

of additives

it

with

of the

as expected

fact,

of random

to the system,

In these

formation

no cooperativity

by a poly-L-lysine-heme

as ligand

reaction

coordination

structure

and in

polymer-ligands

oxygen

axial

no conformational

the effects

the

a disappearence

in a poly-4-vinylpyridine

with

of the

occur

that

reaction.

we studied

molecular

shown

may not

concerning

Nextly,

do not

the

change

by the

in

However,

the complex

such a dynamical

that

a cooperative-binding

conformation. system,

structure

resulted

1,may initiate

regular

polyethyleneimine

caused

which

RESEARCH COMMUNICATIONS

(3),indicated

of a helical

oxygen,

number

with

so that

poly-L-lysine

of the strain

or molecular

AND BIOPHYSICAL

;

Vol. 67, No. 3, 1975

Table

2

BIOCHEMICAL

Effects

oxygen

of additives

on the

by poly-L-lysine-heme

Additives

2.0

130

polyethyleneglycole

1.3

62

polyglutamic

acid

1.7

80

sodium

chloride(O.lM)

1.7

14U

sodium

chloride(0.4M)

1.3

270

complex

was not affected

in a cooperative helical

shows that

at all

parameter

structure

tion

which

the

n 2.0

2.5~10~

2.0

4.6~10~

structure

of deoxy-heme

Therefore,

the decrease

by the instabilization

which

soln.

K(20°C)(M-2)

polymers.

may be caused

of molecular

at 30°C in PH 12 buffer

overall

by additive

of poly-L-lysine,

behavior

P50(mmHg)

None

polymers,

RESEARCH COMMUNICATIONS

absorption

complex

Cooperative parameter (n)

of additive

of an c1-

may mean a destruction

of an informa-

transfer. On the

other

monoxide ference

hand,when

or molecular

cause

potassium

cyanide

lysine-heme

Then,

constant

the

parameter

stability

structure

to cause

increase

of molecule species.

because

the

a ligand-exchange than

those

of the concentration while

salt

of sodium

chloride

of a heme complex.

can be explained

Generally,

in an aqueous

it solution

868

oxygen

the

is well becomes

a poly-Land carbon

absorption

complex.

Therefore,

due

of

with

on the

coordination

increased.

is,

difbut

concentration

of sodium

the axial

constant

That

of molecular

of neutral

This

absorbed

reaction

by a poly-L-lysine-heme

decreased,

of polypeptides

in size

strength

of carbon

was observed.

and an ionic

the effect

oxygen

and the formation

P50 by the addition

difference

ionic

we studied

was used in place

no cooperative-binding

was much higher

of molecular 2, with

erative

enough

cyanide

a neutral

may be the

complex

monoxide.

Table

oxygen,

between

essential

behavior

potassium

may not be due to the

to the difference

the

AND BIOPHYSICAL

As shown

chloride, number the

a coopwas kept

increase

by the

increase

known that instable

in

of in

a helical by adding

Vol.67,No.3,1975

sodium

chloride.

explained ligand

So the

by the

decrease

instabilization

AND BIOPHYSICAL

in a cooperative

RESEARCH COMMUNICATIONS

parameter

of an a-helical

observed

structure

may be

of the polymer-

by salt. This

from

system

consisting

the standpoint

studying It

BIOCHEMICAL

wiil

of poly-L-lysine

of a synthetic

factors

by both

be reported

in

Acnowledgment

The authors

For a Grant-in-Aid

model

experimental the near

and heme is of cooperative

and theoretical

extremely systems.

methods

interest We are now

of approach.

future. wish

to thank

the Ministry

of Education,

Japan,

(No. 911503).

-Rei 'erences 1.

Tsuchida,E.,

Honda,K.,

and Sata,H.

(1974)

Biopolymers

2.

Tsuchida,E.,

Honda,K.

, and Sata,H.

(1975)

Polymer

3.

Tsuchida,E.,

Honda,K.

, and Hasegawa,E.

(1975)

-13 2147-2159 J. L 498-504

Biochim.

Biophys.

Acta

393 483-495 4.

Willstaetter,R.

5.

Miller,J.R.

6.

For example: et al eds.) Tsuchida,E.,

(1950)

Ann.

and Dorough,C.D. Wyman,J. Vol.

19,

Honda,K.

(1964)

Chem. 365 197-202 (1952)

3. Amer.

in Advances

pp. 223-286,

Academic

, and Hasegawa,E.

in contribution

869

Chem. Sot.

in

-74 3977-3981 Chemistry (Anfinsen,C.B

Protein Press,

(1975)

New York

Biochim.

Biophys.

Acta