Optical rotatory dispersion studies of the heat denaturation of avidin and the avidin-biotin complex

Optical rotatory dispersion studies of the heat denaturation of avidin and the avidin-biotin complex

BIOCHEMICAL Vol. 25, No. 5, 1966 AND BIOPHYSICAL RESEARCH COMMUNICATIONS OPTICAL ROTATORYDISPERSION STUDIES OF THE HEAT DFJVATURATION OF AVIDIN AND...

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BIOCHEMICAL

Vol. 25, No. 5, 1966

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OPTICAL ROTATORYDISPERSION STUDIES OF THE HEAT DFJVATURATION OF AVIDIN AND TRE AVIDIN-BIOTIN Alan B. Pritchard,

Donald B. McCormick,

[email protected]* and Lemuel D. Wright

Graduate School of Nutrition and Biochemistry Section, Division of Biological. Sciences, Cornell University, Ithaca, New York

Received October 31, 1966

Avidin, biotin

a protein

to yield

(Green, 1963a). stable

isolated

a complex with a dissociation The avidin-biotin

toward proteolytic

pH range (Eakin, 1942).

Snell,

of biotin

and Willisms, solutions

1964; Pai and Lichstein,

exhibits

and positive visible

at 285 mp.

region.

complexing

prominent

from its

complex

but the increased

of high ionic

strength

affinity affords

the complex with heat (Wei and

(Green and Melsmed, 1966), rotatory

Cotton effects

dispersion

the

(ORD) spectrum

which are negative

The curve approximates

The ORD spectrum of avidin

with biotin,

biotin

1964).

study shows that the optical.

of avidin

l*O),

toward disrupting

In agreement with a recent report present

as 10 -15 M

reported

1940; GyUrgy, Rose, and Tomsrelli,

was shown to release

in buffer

protection

constant

with

complex has been shown to be relatively

and Williams,

Snell,

for avidin

considerable Wright,

(Eakin,

combines firmly

enzymes (Gyisrgy and Rose, 1943) and over a wide

Steam sterilization

with avidin

from egg white,

zero rotation

is not markedly

at 245 ml.r into

changed upon

and only moderate changes occur in 8 M urea.

*This work was supported by N.I.H.

Grant No. AM-08721.

524

the

BIOCHEMICAL

Vol. 25, No. 5, 1966

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

However, we have now found that heating causes a precipitous

reversible

of avidin

above 80'

below 330 mn reflecting

increase in levorotation

denaturation

solutions

which can be prevented by complexing

ir-

with bioti.n.

Experimental CrystsLiine Biochemicsls

D-biotin

Corp.

and crude avidin

The avidin

carboxymethyl-cellulose (McCormick,

The ORD measurements were obtained with a thermostated

Spectra were determined temperatures of avidin without

by chromatography

on

(Melsmed and Green, 1963) and biotin-cellulose

1965).

Spectropolarkneter

was purified

were purchased from Nutritional

at room temperature

as specified. (0.5 mg/ml)

urea or biotin

Solutions

compartment and 2.5 cm cells. or after 15 minutes

contained

in Nf$OH-NHbC1 buffer

a constant

at such

concentration

(pH p, n = 1) tith

and

as specified. Results

The ORD spectra of native 8 M urea, and heat-denatured

+200-

cell

using a Cery 60

and Discussion avidin,

avidin

avidin-biotin are shown in Fig.

complex, avidin 1.

A

I 250

I 270

290

I 310

x w

Fig. 1. ORD spectra of avidin in buffer (o), with 2.9 x 10V5 M biotin (A), in 8 M urea (o), and when heated at 85O (0).

525

with

Vol. 25, No. 5, 1966

Avidin

BIOCHEMICAL

in distilled

exhibits

AND BIOPHYSICAL

water or buffer

a maximal positive

favorable

rotation

and a maximel negative rotation

at 260 mu.

from those of many globular

Cotton effects

in the ultraviolet

a slight

intensification

proteins,

Inclusion

rotations

complexing

of tryptophan

by N-brqPaosuccinimiae

sunewhat diminishes

260 w, but concentrations reported

to cause little

prolonged

of biotin

treatment

avidin tions

will

probably avidin

Also earlier

including

Addition

of

at both 295 and

hydrochloride

As with

(Green and Melsmed,

rotation

at

at wavelengths below 330 mp.

heated solution

avidin

does not

at room temperature

work has shown that such heat-denatured (Wei

and Wright,

at 85') may disrupt

those involved

1964).

secondary bonding

in the sub-unit

structure

The condiforces, reported

for

(Green, 183~). The heat denaturation

changes in specific The structure

of avidln

rotation

of avidin

temperature,

rapid disruption

BY 85O, extensive

bind biotin

can be followed

at either

remains stable

evidenced by lack of significant

so".

(Green, lg63b).

as the spectrum of native

not complex with biotin

used (15 minutes

impedes oxidation

as high as 9 M have been

in levorotation

reappear upon keeping a previously

elicits

at both 295 and 260 mp.

causes ccmpl.ete loss of the positive

increase

of

from

of avidin (Green, 1963~).

with 6 M gusnidine

These changes are irreversible,

for many hours.

in the solution

by avidin

solute

spectrum is some-

contributions

the dispersion

clenaturation

1966), heat denaturation 295 ~I.L and a large

of this

at 287 w

high in I,-tryptophan

of biotin

It has been shown that

8 M urea to avidin

of biotin

but the positions

reflect

reportedly

of specific

residues

The overall

expectedly

amino acid composition,

(Melamed and Green, 1963).

for binding

at 295 w with a shoulder

what different

the aromatic

RESEARCH COMMUNICATIONS

readily

295 or 260 m/~ as shown in Fig. 2. at temperatures

below 70" as

change in the ORD spectrum. of structure

loss of the original.

are found.

526

by the

Above this

occurs with an apparent structure

end ability

'Ihr of to

BIOCHEMICAL

Vol. 25, No. 5, 1966

The effect of avidin

concentration

is shown in Fig.

protection

of avidin

irreversibly required

of biotin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3.

on preventing

All concentrations

of biotin

when heated at a temperature

denature this

protein

for a 1:1molecuJer

alone.

heat denaturation effect

(85’) sufficient

Increasing

the level

to to that

ccmplex affords maximum protection,

higher

concentrations

of biotin

biotin

complex remains stable

do not alter until

this

temperatures

some

situation.

and The avidin-

near that of boiling

water are reached. Fig.

2

Fig.

3

+300-

0

o-

p-,"-" X 295

ol ‘0 x

-3oo-

70

-200

t 0/

my

,

2. -500

-

-900

-

-6OOv I 30

I 50

‘C

I 70

8 90

d

0

1

1

2.0 Moles

I

I

I

4.0 BiotinlMole

I

60 Avidin

Fig. 2. Heat denaturation of avidin as measured by ORB changes at two wavelengths. Fig. 3. Effect of biotin concentration on preventing the heat denaturation of avidin at 85O as measured by ORB changes at two wavelengths.

Acknowledgments We wish to express our appreciation Scheraga for providing

to John Vournakis

and Harold

equipment and assistance.

References Eahin,

R. E., gnell,

801 (@a).

E. E., and Williams,

Green, N. MO, Biochem. J.,, 89, 585 (lsja). Green, N. M., Biochem. J., 3, 5gg (lg63b).

527

R. J., J. Biol.

Ghan., l&

Vol. 25, No. 5, 1966

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

Green, N. M., Biochem. J., B, 609 (1963~). Green, N. M., and Melsmed, M. D., Biochem. J., 100, 614 (1966). Gyi%v, P*, and Rose, C. S., Proc. Sot. Exptl. xl. Med., 2, 55 (1943). Gyijrfg, P., Rose, C. S., and !I!omarUi, R., proc. Sot. Exptl. Biol. Med., 3, 169 (1942). McComlck, D. B., Anal. Biochem., 13, 194 (1965). Melsmed, M. D., and Green, N. M., xochem. J., 2, 591 (1963). Pai, C. H., and Lichstein, H. C., Proc. Sot. Exptl. Biol. Med., -~6, 197 (1964). Wei, R. D., and Wright, L. I)., Proc. Sot. Exptl. Biol. Med., 117, 341 0964).

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