Enzymatic synthesis of malonaldehyde

Vol. 82, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Moy 30,1978

Pages

547-552

ENZYMATIC SYNTHESIS OF MALONALDEHYDE Frank

W. Summerfield

Department University

'Received

April

and Al L. Tappel

of Food Science and Technology of California, Davis, CA 95616

4,1978

SUMMARY Malonaldehyde was prepared from 1,3-propanediol by alcohol dehydrogenase. The Km for 1,3-propanediol was about 1.7 mM. The reaction proceeded best at low ionic strength and at pH 9. The reaction was unaffected bY2pyro$psE;;y, phosphate, bicarbonate, or N-ethylmorpholine buffers, or by Mg , Ca or citrate. However, the reaction was inhibited 50% by 1.5 mM borate, 1 mM ' cyanide, and 5 mM azide. Thiols, such as dithioerythritol, inhibited the reaction 50% at 50-100 PM, while others, such as mercaptoacetate, inhibited 50% at concentrations over 1 ti. Malonaldehyde was removed from the reaction mixture by evaporation at pH 3 and condensation at -78°C. No other products associated with lipid peroxidation were produced. The method was useful for preparation of radiolabeled malonaldehyde. INTRODUCTION Malonaldehyde radiation

damage

lipids

(4),

actions Until acidic

it

concentration radiolabeled

in aging

and nucleic

malonaldehyde

was necessary

hydrolysis

protonated

(5),

involve

implicated

(l),

acids

(6).

to prepare

(8).

In either

of malonaldehyde tetraalkoxypropanes

For these

reasons,

a new synthesis

MATERIALS

AND METHODS

are

rapid

(Z),

groups

the study

by the (7)

a few millimolar. available,

tracer

occurs Also,

studies

of reareas.

high-temperature,

or by shaking

polymerization

and

on phospho-

in many biochemical

malonaldehyde

case, exceeds

amino Therefore,

is of interest

of a 1,1,3,3-tetraalkoxypropane

resins

carcinogenesis

It has been shown to crosslink

(3).

proteins

that now,

has been

since cannot

with if

the no be done.

was developed.

Equine liver alcohol dehydrogenase (EC l.l.l.l), rabbit muscle lactic dehydrogenase (EC 1.1.1.271, and NAD were obtained from Sigma Chemical Co.; and citrate from Mallinckrodt sodium phosphate, pyrophosphate, bicarbonate, Chemical Works; sodium pyruvate and N-ethylmorpholine from Nutritional Biochemicals Corp.; and 1,3-propanediol and 1,1,3,3-tetramethoxypropane from Eastman Organic Chemicals. The 1,3-propanediol was distilled twice, and only the fraction boiling above 200°C was used.

0006-291X/78/0822-0547$01.00/0 547

Copyright All rights

0 1978 by Academic of reproduction in any

Press, Inc.

form reserved

BIOCHEMICAL

Vol. 82, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

01 FIGURE 1. Time

course

for

FIGURE 2. Hanes-Woolf centration.

production

plot

of malonaldehyde.

of enzyme activity

as a function

of substrate

con-

Unless otherwise stated, all reactions were carried out at 25'C in 50 mM pyrophosphate buffer, pH 9, with 0.0015 unit (1 unit is equivalent to 1 pmole product/min) of alcohol dehydrogenase, 0.005 unit of lactic dehydrogenase, 50 M NAD, 1 mM 1,3-propanediol, and 20 r&l pyru te in a final volume of 1 ml. [l- Y4C]-1,3-Propanediol for the preparation of Y3C-malonaldehyde was obtained from ICN Chemical and Radioisotope Division. Malonaldehyde was determined by a modification of the thiobarbituric acid reaction (9). The reaction mixture, or an appropriate aliquot thereof diluted to 1 ml, was mixed with 1 ml of glacial acetic acid and 3 ml of 10 mM thiobarbituric acid and the mixture was heated in a covered test tube in a boiling water bath for 1 hr. Tetramethoxypropane was used as a standard, and the absorbance was read at 532 nm. RESULTS The time lag rate not

of about

increase

20 min,

beyond

mM 1,3-propanediol. nase for

by higher

about

was the

The reaction of the

at about

After

an initial

50 nanomoles/hr.

temperatures,

of 1,3-propanediol was allowed

initial this

lag. value

alcohols

is

but

The

the yield

The plot large,

dehydrogenase

548

concentration

to proceed

the

is much greater

the NAD requirement

Kd of alcohol

1.

did

65%. the effect

Though

3 displays

shown in Figure

was increased

many non-biological

Figure

is

was formed

2 illustrates

the effect

reaction

malonaldehyde

of the reaction.

minimizing

factor

of the

of the reaction

Figure rate

course

reveals

4 hr,

than

NAD, about

thus

a Km of about

Km of alcohol

of the reaction. for

for

on the

for

1.7

dehydroge-

ethanol

(10).

The determining 30 uM (11).

Vol. 82, No. 2, 1978

BIOCHEMICAL

LOG NAD,

FIGURE 3. Dependency

of reaction

ity

4 depicts

was observed

The left

side

optimum

activity

Figure pyruvate ionic

be increased

0.1.

activity

because

lactic

activ-

at pH 8 or 10.

dehydrogenase

has

pH 7.5. of ionic

strength Thus,

of the

N-ethylmorpholine,

strength

on the reaction.

Here,

the

was used so that

could

be used.

The results

showed

no maximum

above

0.02,

substantial

activity

at ionic

for

reaction

phosphate,

50% inhibition

a 50% inhibition EIITA had little erythritol

optimum

but

good activity,

the

ionic

strength

should

not

unnecessarily.

The rate

caused

significant

may be steeper

as low as 0.02

below

Though

was 10 mM, and 10 mM N-ethylmorpholine

at any ionic

strengths

was still

the effect

concentration

activity

others,

curve

of pH.

of pH on the reaction.

at pH 9, there

5 shows

on NAD concentration.

as a function

at about

strengths

rate

the effect

of the

RESEARCH COMMUNICATIONS

pM

FIGURE 4. Enzyme activity

Figure

AND BIOPHYSICAL

was not

or bicarbonate

by affecting by 1 n$l cyanide

effect.

Thiols

and mercaptoethanol, such as cysteine,

significantly

required

the

changed

buffers;

lactic

caused

50% inhibition

2-5 mM concentration

549

groups,

use of

1.5 mM borate There

but Ca +2, Mgt2,

hydroxyl

tion.

however,

dehydrogenase.

and 5 mM azide, containing

by the

was also

citrate,

such as dithio-

at 50-100 to cause

PM, while 50% inhibi-

and

Vol. 82, No.2,

1978

05

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

06

LOG IONIC STRENGTH

FIGURE 5. Effect

of ionic

FIGURE 6. Effect hyde.

of pH on the

Kwon and Watts of malonaldehyde affected

(7)

proceeded 5O'C.

from for

with

spectral

a pK of about

malonaldehyde

4.5.

24 h, the

By this

obtained.

pH.

absorption

the

Since

changes With

were

this

The solution

its

When the

removal

reaction

stream

and then

of had to

condensed

by evaporation

of up to 40 tnM malonaldehyde stored

Figure

to pH 3 and heated

to pH 7 and concentrated

if

is

pK.

a scheme for

was adjusted

concentrations

spectrum

of malonaldehyde

used to determine

by an air

was adjusted

was stable

volatility

was devised.

mixture

of malonalde-

absorption

information,

was volatilized

method,

spectrum

ultraviolet

the

mixture

reaction

The condensate

at 35°C.

ultraviolet

the reaction

The malonaldehyde

at -78'C.

on enzyme activity.

have shown that

changes

by pH, these

6 reveals

strength

PH

were

at pH 7 and 4°C.

DISCUSSION Alcohol

dehydrogenase

corresponding since

only

(10).

malonaldehyde

steady-state reaction

aldehydes

converts

thermodynamic the malonaldehyde

variety

study,

the

an initial

of alcohols

However,

was reacting

over it with

90% conversion is quite itself

550

to their

substrate

was a diol,

and

lag

be expected

for

of hydroxypropionaldehyde

equilibrium,

basis.

In this

was detected,

concentration reached

a wide

would

to be reached. would

possible as rapidly

If the

be expected

that

at this

as it

a

on a

concentration,

was formed.

Since

Vol. 82, No. 2, 1978

higher

substrate

is

likely

not

concentrations that

The study system with in

for

most

it

is

buffers,

salt

system

described

micelles

hydrogen

must be used, products

useful

peroxide

described

no peroxides

or biochemical

commercially thus

Therefore,

polyunsaturated

low,

the

Malonaldefatty

However, very

do

bacteri-

of investigations.

is

than

different

times.

or lipoxygenase.

acid

oxygen

or per-

and many other

available

possible

[l-

to prepare

Thus,

products

Finally, 1

necessary,

or ultraviolet

secondary

damage

if

products.

ac d reaction

can be used to prepare

is

present,

or thiols;

reaction

a variety

reactive

or other

and also

it

resembles

may require

can be used anaerobically

as a thiobarbituric

from

long

by a

the molecule

is

chelators,

of malonaldehyde

prepared

toxicity,

contains

concentration

by treating

(6)

the yield

for

be facilitated

more closely

cations,

for

in situ

would

molecules

it

[l-l4

malonaldehyde absorption

related

rancidity, was prepared

by the methods malonaldehyde

for

can be standard

to food

Cl-malonaldehyde

4C]-1,3-propanediol

radiolabelled

and it

described;

tracer

studies. ACKNOWLEDGMENT This National

re-

can be formed.

The system generates

low

target

may be necessary

be generated

active

a constant,

concentrations,

enzyme.

that

of system

divalent

may prove

with

type

Different

hyde can also

oxide

mixture

concentrations,

such as azide

the

in a reaction This

RESEARCH COMMUNICATIONS

product

was inactivating malonaldehyde

in which

systems.

in higher

involving

to react.

conditions,

in vitro

tides

malonaldehyde

generation

AND BIOPHYSICAL

resulted

of reactions

its

which vivo

BIOCHEMICAL

research Institute

was supported

by NIH research

of Arthritis,

Metabolism

grant

AM 09933

and Digestive

from the

Diseases.

REFERENCES 1.

Munkres,

K. D.

(1976)

2.

Kubinski, (1973)

3.

Packer, L., Deamer, Res. 2, 23-64.

Mech.

Aging

M. A., H., Strangstalin, Can. Res. 33, 3103-3107.

Devel. Baird,

D. W., and Heath,

551

5, 171-191. W. M.,

R. L.

and Bountwell,

(1967)

Advan.

R. K. Gerontol.

Vol. 82, No. 2, 1978

8lOCHEMlCAL

4.

Bidlack,

W. R.,

5.

Chio,

6.

Reiss, U., Chio, K. S., Commun. 48, 921-925.

7.

Kwon,

8.

Brooks,

9.

Wills,

K. S.,

T.,

10.

Dickinson,

11.

Theorell, 1114.

and Tappel,

and Tappel,

and Watts, R. B.,

E. D.

F. M., H.,

A. L. A. L.

(1973)

(1969)

and Tappel,

8. M.

and Klamerth, (1966)

AND BIOPHYSICAL

Biochem.

and Dalziel,

and Bonnichsen,

Biochemistry

R. K.

552

8, 2827-2832.

(1972)

Biochem. 28,

(1968)

J. Biochem.

99,

K.

8, 203-207.

J. Food Sci.

0. L. J.

Lipids

A. L.

(1963)

RESEARCH COMMUNICATIONS

Eur.

Biophys.

Res.

627-630. 5, 178-182.

667-676.

(1967) (1951)

Biochem. Acta

J.

104,

165-172.

Chem. Stand.

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