Ascorbic acid interaction with metallothionein

Vol. 41, No. 5, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ASCORBIC ACID INTERACTION G.W. Evans,

P.F.

WITH METALLOTHIONEIN

Majors

and W.E.

Cornatzer

Guy and Bertha Ireland Research Laboratory, Department of Biochemistry, University of North Dakota Medical School, Grand Forks North Dakota 58201

Received

October

26, 1970 SUMMARY

Analysis of metal binding by metallothionein demonstrated that ascorbic acid decreases binding of cadmium, copper and zinc by metallothionein from both bovine duodenum and liver. Spectral analysis of metallothionein following addition of ascorbic acid indicated that ascorbic acid interacts with metallothionein. These results suggest that ascorbic acid alters the metabolism of cadmium, copper and zinc by competing for sulfhydryl bindings sites on metallothionein. Ascorbic Dietary

ascorbic

deficiency that

acid

acid

in chicks

control

decreased dietary

affects

diets

and cadmium

acid

Fry

(5)

cadmium

demonstrated toxicity

In a recent metallothionein protein,

alters

Hill

acid

isolated that

with

severity

Hunt --et al.

ascorbic

acid

and Starcher the

(2)

hepatic

decreases

supplemental

rat.

dietary

(3)

observed in

demonstrated

that

of either

orally

et al.

the uptake

from

of copper

resulted

uptake

64cu . Sahagian

intestine

elements.

(4)

of both

Recently, ascorbic

zinc

Fox and acid

prevents

in the quail. publication, in bovine

metallothionein,

Therefore, acid

the

the

Furthermore,

decreases

ascorbic

of several

increase

administered

that in

(l-3).

copper.

or intraperitoneally demonstrated

supplements

supplemented

hepatic ascorbic

the metabolism

attempting

we demonstrated duodenum binds

to explain

the metabolism

and liver

cadmium,

zinc

the mechanism

of these

elements,

1244

the presence (6).

of

The sulfhydryl-rich

and copper

(7-9).

whereby

ascorbic

we have

examined

the

BIOCHEMICAL

Vol. 41, No. 5, 1970

interaction

between

ascorbic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

acid

and purified

netallothionein.

MHTHODS Metallothionein liver

was purified

by the method

dissolved were

in

10 -3 M Tris

adjusted

the

metallothionein, or 2

2 mixed

stirred

duodenum

Ascorbic

acid

to pH 8.6.

and

was

Radioactive

isotopes

of ascorbic

acid

on metal

binding

by

1.0 x 10D2 M ascorbic

acid

and 1.6 x 10e5 M

l

metallothionein

dialyzed

for

dialyzed

fractions

10 -3 M Tris,

in for

24 hours

scintillation

2 hours

against

were

counter

at 4 C after

two changes

assayed

for

and protein

The solutions

pH 8.6. which

the

of distilled

radioactivity

were

contents

were

water.

The

in a gamma well

was determined

using

biuret

(10). To analyze

the

and adjusted

effect

continuously

reagent

(9).

bovine

or x 10 -5 M 65ZnC1 x lO-5 M 64 Cu 2.14 2 25 bJ’33)2 -4 10 with 1.52 x M hepatic metallothionein or 3.8 x 10 -5 M

115mCdCl

duodenal

et al.

both

to pH 7.0.

To analyze

were

of Pulido

from

the

ultraviolet

absorbance

spectrum

spectrophotometer,

Tris , pH 8.6.

Thereafter,

pH 8.6, was added

Tris,

of ascorbic

in 10 -3 M Tris,

metallothionein recording

interaction

at room temperature and corrected

for

with

was determined

The reference

cuvette

0.1 ml of 10m3 M ascorbic to both

metallothionein,

x 10 -5 M hepatic

of 3.16

pH 8.6,

incubated

cuvettes,

and a second volume

acid

absorbance

spectrum

on a Cary

10 -3 M

contained acid for

10 -3 M

in

10 minutes

was determined

change.

RESULTS AND DISCUSSION The results inhibits

metal

ascorbic

acid

cadmium,

copper

Cadmium binding

shown in Table binding

by metallothionein.

significantly and zinc

1 demonstrate

decreased by both

was the least

1245

ascorbic

The addition (P < .05) the

duodenal

affected

that

and hepatic

by ascorbic

acid of

binding

of

netallothionein. acid,

15

which

is

BIOCHEMICAL

Vol. 41, No. 5,197O

Table

1.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The effect of ascorbic acid on metal duodenal and hepatic metallothionein.

Duodenum metallothionein Additions

115mCd + ascorbic

64Cu + ascorbic

acid

65Zn 65 Zn + ascorbic

probably

acid

shown

28,741&g

6,85o+log

24,26g-t511

4o,g22+1,852

200,61g+g,438

25,618+g21

95,521+2,604

20,025?370

121,243+2,462

14,182+27g

43,945+'+58

are mean + S.E.

due to the

high

Addition

of ascorbic

the ultraviolet

absorption

of three

binding acid

CPM/mg

7,505+126 acid

64CU

by

Hepatic metallothionein

CWmg

ll!jrncd

Values

binding

experiments.

constant

for

cadmium

to metallothionein

spectrum

(8).

markedly

of the protein

altered

(Figure

1).

I 0.6

0.4 8 ,z lg a $

-

+ ASCORBIC

ACID

0.3 -

0.2 -

0.1 -

240

250

260 WAVELENGTH

Figure hepatic

1.

Ultraviolet

metallothionein. 1246

270

260

290

( mp)

absorption

spectra

of

BIOCHEMICAL

Vol. 41, No, 5, 1970

Metallothionein

contains

the metal Since

ions

in the

ascorbic

and alters probably

acid

the

for

these

a complex

metals.

metallothionein.

zinc

probably

ascorbic

with

described

here

acid

of cadmium,

copper

formation

metal

binding

the

for

mediates

the

intestinal

the

necessary

probably

exclusively

that

doubt

binding

sites

properties

from However,

the

absorption

absorption

the sulfhydryl-binding

metallothionein.

is without

in metallothionein.

inhibits

of the chelating

interaction a factor

(7-g).

vitamin

groups

in the metabolism result

the

thiol

acid

and

by metallothionein

spectrum,

Ascorbic

demonstrate

and ascorbic

by mercaptide

by providing

do not with

bound

and zinc

the alterations

acid

groups

absorption

In view

(11)

of sulfhydryl

are

inhibits

by competing

acid

content

apparently

copper

the elements

protein

both

Metallothionein of cadmium,

a high

ultraviolet

forms

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of on

of ascorbic

of cadmium, the

sites

copper

interaction

and of

the experiments between affecting

metallothionein the homeostasis

and zinc.

REFERENCES

1.

2. 3. 4.

2: 2 9: 15. 11.

Carlton, W.W., and Henderson, W., J. Nutr., &, 67 (1965). B., J. Nutr., &, 271 (1965). Hill, C.H., and Starcher, J., and Newberne, P.M., Br. J. Nutr., Hunt, C.E., Landesman, 24, 607 (1970). Sahagian, B.M., Harding-Barlow, I., and Perry, H.M., Jr., J. NutrM i>ss Fox 291 (Wi?. . D ., and Fry, B.E., Jr., Science, lf& 989 (1970). Evahs, G.W., Majors, P.F., and Cornatzer, W.E., Biochem. Biophys. Res. Commun., 40, 1142 (1970). Kagi, J.H.R., and Vallee, B.L., J. Biol. Chem., a, 3460 (1960). and Vallee, B.L., J. Biol. Chem., a, 2435 (1961). Kagi, J.H.R., Pulido, P., Kagi, J.H.R., and Vallee, B.L., Biochemistry, 2,

1768 ( 1966). Gornall, A.G., Bardawill, C.S., and David, M.M,, J. Biol. iiz, 751 (1949). Hopping, J.M., and Ruliffson, W.S., Am. J. Physiol., 210, (1966)

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