Streptavidin—A substance with avidin-like properties produced by microorganisms

Streptavidin—A substance with avidin-like properties produced by microorganisms

Vol. 14, No. 3, 1964 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS STREPTAVIDIN-ASUBSTANCEWI!I'HAVIDIN-LIKE PROF!EXl'IESPRODUCED BY MICROORGA...

266KB Sizes 0 Downloads 45 Views

Vol. 14, No. 3, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

STREPTAVIDIN-ASUBSTANCEWI!I'HAVIDIN-LIKE PROF!EXl'IESPRODUCED BY MICROORGANISMS Fred Tausig and Frank J. Wolf Merck Sharp & DohmeResearch Laboratories Division of Merck and Co., Inc. Rahway, NewJersey

Received

October

30, 1963

Animal dietary

studies demonstrating the toxic

white (Boaz, 1924), and the recognition foods and extracts

identity toxic

of raw egg

action of certain

(Boaz, 1927) led to the

1931, 1939) and subsequent proof of the latter's

with the yeast growth factor

factor

of the protective

against this egg white injury

work on Vitamin H (Gyzrgy,

effect

biotin

of egg white, a heat labile

(Gygrgy et al.,

protein,

was first

1940).

The

isolated by

Eakin and coworkers (Eakin, Snell and Williams,

1940, 1941), and named

avidin.

by binding biotin

firm,

It was shown to exert its toxic non-digestible

effect

cconplex and thus preventing the utilization

vitamin by both animals and microorganisms.

Raw egg white,

avidin

obtained from this source, has since been used

highly

specific

deficiencies

and selective

biotin-binding

into a of the

or purified

extensively

as a

agent to induce biotin

in animals and man, as well as in biotin

requiring

micro-

organisms and isolated enzyme systems. Despite the numerous subsequent investigations biotin

and biotin

antagonists,

avidin or avidin-like

in the field

activity

has thus far

bee- reported to occur only in egg white of birds (Hertz and Sebrell, Jones and Briggs, 1942),

and

l962), the egg jelly

of

1942;

of the frog (Hertz and Sebrell,

in the albumen secreting oviduct tissues of laying hens (Fraps,

Hertz and Sebrell,

1943).

The oviduct tissues of non-laying hens and

mucosal scrapings from the oviducts of pigs, cows and guinea pigs were found devoid of avidin

(Hertz,

1946).

To date, no other source for avidin 205

BIOCHEMICAL

Vol. 14, No. 3, 1964

or truly

avidin-like

binding activity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

materials has been reported.

0.2 percent that of avidin

of pure salinine,

Longsworth, 1942) and of bull

The very slight biotin(Wooley and

sperm, 0.2 percent that of hen egg white

(Jones and Briggs, 1962), is of such a low order that non-specific rather

than true avidin activity,

binding,

may be implicated.

The present paper presents evtilence for the elaboration tomycetes of a protein with biotin-binding

properties

similar

by Strep-

to avidin.

This material was encountered during a search for new antibiotics several different

types of Streptomycetes (Stapley et al.,

to produce antibiotic

substances primarily

Other, more detailed

bacteria.

active

report8 will

in which

1963) were found

against gram-negative

describe the antibacterial

and

other aspects of these substances, such as isolation

and purification

(Chaiet et al.,

and mode of action.

1963), animal studies (Miller,

1963)

Current Investigations During the course of purification was found that the fermentation components; a dialysable, dialysable, mixture

of the antibiotic

broths contained two different

small component (labelled

large component (labelled

"L").

9")

resulted

in cauplete loss of antibacterial

of the two fractions,

at very Ligh levels,

against the samestrain

medium, whereas the "L" fraction It

Was

demonstrated

that

(see Table I).

activity

by itself

as deter-

even

1963).

was found to be

when assayed in synthetic

remained devoid of antibacterial

to the reversal

on

was restored upon

(Chaiet et al.,

of E_. 5

that the difference

could be attributed biotin

'5" fraction

and a non-

one of which was inactive,

when assayed separately

However, the purified highly active

either

kinds of

or gel-filtration

mined in the usual complex assay medium. Full activity recaubination

it

Separation of the bioactive

of the two cauponents by exhaustive dialysis

Sephadex ~-25

activity

in activity

of the inhibitory

activity.

of "S" h the two media properties

of 53" by

The reversal was evident in both media and indicated

"L" acted as a biotin-binding

agent, analogous to avidin. 206

Vol. 14, No. 3, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I ACTIVITY OF '3" AGAINSTE. COLI IN SY'KPHETICMEDIUM AND l!l!S REVERSALBYBIUI!IN

"S"

Inhibition Zone mm.

‘3”

(y/ml.) 40

E

10 10 10 10

a

1.6 032 .064

Biotin (Y-b..)

(l-b-.>

38 33 28

Inhibition Zone mm. 45

0

0

-5

.05 .005 .OOO>

10

35-45, 35-45,

grOOwth

to

The medium contained only glucose, citrate, methionine and inorganic salts, plus agar. Paper disks, 13 mm. diameter, were used saturated with the test solutions. This relationship firmed by the use of purified found to be identical antibacterial

of "L" to the egg white injury avidinl.

The known biotin-binding

to "L" in its ability

properties

in natural

factor

results were obtained using tube dilution

was conagent was

to permit '3" to exert

environments (see Table II).

its Analogous

assays.

TABIX II SlMILARm

cow.

IN EFFEKXOF "L" ANDAVlDIN VERSUSBIUIXN

of each ccqonent,

"S"

"L"

200 100 100 100 100

50:

100 22

33; 333 333

66 66

50

0 0

0 0 0

Avidin

Biotin

0 0

0 0 0 0 0

50:

50 5

0 19

0 14 13

0 0 0 18 0 0 14

3: 3.3 .33 33 3.3 .33

0 33; 333 333

The assay mediumwas made up of Difco nutrient 1 Obtained from Nutritional

E. coli Inhibition Zone ml.

Fn r/ml.

Biochemical Corp. 207

agar plus yeast extract. Activity

- 2,300

35

growth to 25

units

per

@IL

Vol. 14, No. 3, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Animal experiments, reported elsewhere in this journal Tausig, 1963), not only provided further avidin and this new protein first

evidence for the similarity

administered avidin.

Furthermore, this is the first

and

of

from microorganisms, but also constitute

demonstration of the rapid binding of free tissue biotin

terally

(Miller

the

by parenclear-cut

use

of avidin as an adjunct in chemotherapy. Purified

"L" preparations

to those of avidin. biotin

per lwr

exhibit

The biotin-binding

"L".

capacity

The non-dialysable

troyed by heating with the liberation and physical properties fication,

will

many properties

is approximately

protein-biotin

of biotin

closely similar ly of

ccanplex is des-

activity.

The chemical

of 'IL", as well as the methods used for its puri-

be presented elsewhere (Chaiet 5 s.,

1963).

Discussion The finding like activity specific

that microorganisms produce a protein with avidin-

is rather startling,

particularly

in view of the highly

type of action and the fact that until

to occur exclusively amphibia.

in eggs and the oviduct tissues of laying birds and

No explanation

antivitamin

now avidin had been thought

had been found for the presence of this

in these specific

However, it had been suggested that

sources.

avidin plays a role in the physiology of avian reproduction. late was based not only on the restricted the finding

that it

sites of occurrence, but also on

is possible to induce the production of avidin

oviducts of immature and adult non-laying hormones (Hertz,

Fraps and Sebrell,

hens by the administration

in the of

1943, 1944).

Any attempted interpretation

of the significance

duction of avidin by microorganisms is, of necessity, at this time.

This postu-

One plausible explanation

speculative

yould seemto be that it is Dart

of the "defense mechanism" of these antibiotic data show, removal of excess exogenous biotin 208

highly

of the pro-

producing microbes.

As the

from the surrounding environ-

Vol. 14, No. 3, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ment is essential for the activity

of the biotin-reversable

antibiotic

elaborated by these Streptoanycetes. Another possibility

is that biotin-binding

the active enzymes involved in fixing

Excretion

cells.

in detectable

quantities

I.n view of the close similarity avidin

prior to transfer

of carbon

As such these proteins would be present in minute quantities

dioxide. all

biotin

proteins are actually

ana its

isolation

as an appropriate

in

is, however, very unusual.

of this microbial

protein to

fram Streptcanyces, we propose the nameSTREPPAVIDIN

designation for the substance.

References Bcaz, M. A., Biochem. J. 18, 422 (1924). Boaz, M. A., Biochem. J. 21, 712 (1927). T. W., Tausig, F. and Wolf, F. J., Antimicrob. Agents Chaiet, L., Miller, & Chemotherapy, in press (1963). Eakin, R. E., Snell, E. E. and Williams, R. J., J. Biol. Chem. Il.& 801

(1940).

Eakin, R. E., Snell, E. E. and Williams, R. J., J. Biol.

(1941) -

Fraps, R. M., Hertz,

2,

R. and. Sebrell,

W. H.,

140 (1943).

Proc.

Chem. I&,

Sot. Exptl.

Biol.

535 Med.,

Gyorgy, P., Ztschr. f. Arztl. Fortbild.. 28, 377 (1931). Gyorgy, P., J. Biol. Chem. l-& 733 (193g. D. B. and du Vigneaud, v., Gyorgy, P., Rose, C. S., Hofmann, K., Melville, Science 92, 609 (1940). Hertz, R., Physiol. Rev. 6, 479 (1946). Hertz, R. and Sebrell, W. H., Science 96, 257 (1942). Hertz, R., Fraps, R. M. and Sebrell, W. H., Proc. Sot. Exptl. Biol. Med., 2, 142 (1943). Hertz, R., Fraps, R. M. and Sebrell, W. H., Science 100, 35 (1944). Jones, P. D. and Briggs, M. H., Life Sciences 1, 6213962). Miller, A. K., Antimicrob. Agents & Chemotherapy, in press (1963). Miller, A. K., Tausig, F., this issue. Woolley, D. W. and Longsworth, L. G., J. Biol. Chem. 142, 285 (1942).

209