Fluorescence titration of native and PMS-subtilisin Carlsberg

Journol of Molecular Structure. 115 (1984) 285-288 Elsevier Science PublIshem B.V., Amsterdam - Printed In The Netherlands

FLUORESCENCE

TITRATION

R.

BOTCVA

Institute

of

Organic

Bulgarian

Academy

N.

GENOV,

OF

and of

NATIVE

AND

285

P&IS-SLIBTILISIN

CARLSBERG

PI. SHOPOVA

Chemistry

with

Sciences,

Sofia

Centre

of

1040

Phytochemlstri

(Bulgaria)

ABSTRACT The effect of pH on the tyroslne and tryptophan fluorescence of both native and phenylmethanesulfonyl (PXi)-subtllisln CarlsThe observed transltlons In the titration curves berg is studled. at acidic pHs reveal considerable changes in the tnree-dlmensional structure of this enzyme. Conclusions about the conformatlonproieal stabilities of subtlllsln Carlsberg and the homologous ase DY are made.

INTRODUCTION Fluorescence peclally

emission

this

vironment

of

and

serve

The

fluorescence

parameters

nrldth and

the

These

with

conformational

XATERIALS

AND

Subtlllsln fessor

Ib

Carlsberg

Batch

measurements

0.025

effect.

before

using.

rosine

amrde

effect

of

posItIon,

;n zacp5

on

the band

spectral

Carlsberg.

Srmilar

transltlons

esperl-

17 some

ca-

associated

pIis.

prepared

The

native

Quantum

were

05

Chemistry,

ln

ref,l.

of of

described

a Corrected

The

were 0.11

0.13

Eron

orl a PerkIn-Elner

trrs

used.

enzymes

and

Deparinent

as

with

wavelength

yields

(AcTyrNi12)

gift

performed

equipped

excitation

a generods

Laboratory,

were

buffers

the

OO22-2860/84/$03.00

was

$1 phosphate-citrate,

bonate-bicarbonate

filter

t'nelr en-

changes

DY ilere performed

acldlc

DY was

Spectrofluorimeter,

at

maxlmulm

60420

(Carlsberg

Fluorescence

0.05

the

CO

es-

i'lETtiODS

Subtilisin

below

sensitive

fluorescence at

In ?rotelns,

structural

subtillsin

changes

Svendsen

Accessory.

LS

of

protease

revealed

Copenhagen). XPF-44

of

groups

describes

(emlsslon

yield)

studies

a prooe

paper

homologous

ses.

aro;natic

reslddes,

as

present

quantum

with

the

tryptophyl

can

romolecules.

ments

of

optical

purified (ref.2)

(ref.3)

0 1984 Elsevier Science Publishers B.V_

for for

model

Spectrum

h>-drochloride

in order

or

densltles to by

Pro-

avoid

cark'ere

inner

gel-filtration

N-acetyl-L-tyN-acetyl-L-tryp-

2.66 tophan

amide

RESULTS

(AcTrpNH2)

AND

Subtilisln

Carlsberg

a single

Studies

at

due

circular

native

and

ximum band

of

ne dS

14

(AA)

fluorescence

10. 0

(PMS

could

a light

the the

at

(Amax)

at

tyrosyl

seen

performed this

protease

residues with

reason The

Judged

from

275

leads

303-304 is

and

fluorescence DY

tyrosyl

we

per

the

conmolecule.

native

used

en-

phenylme-

modification the

did

fluorescence

not and

nm

at

they

did

not

Carlsberg)

in

Fig.

per

neutral These

to a simultaneous

protease

molecule) pH.

values change

and

residues.

PMS-subtillsin

(homologous

residues

nm.

and

nm

tryptophyl of

31-32

subtrlisin be

bacterial

protease. as

PMS-subtilisin and

width

values.

spectra.

all

conditions this

be For

structure

with

of

tryptophyl

standard

13 tyrosyl

cannot

autolysls.

dichroism

excitation

as

alkaline

and

(PMS)-inhIbited

Irradiation

these

pHs

to a fast

the

is an

tryptophan

alkaline

thanesulfonyl change

chosen

DISCUSSION

taining

zyme

were

Under

Carlsberg containing exhibits

The

respective

are

typical

in

the

5.5-10.0

1

a maspectral

for

tyrosr-

pH-regions

5.5-

(PMS-subtilisin

DY)

1.

E

c

X

x

bE 2

3

4

5

6

9

10 PH

) and of pH on the spectral maximum position (A Fig. 1. Effect the spectral band width (no) of native and PMS-subtilisi%XCarls. Carlsberg; (A-A)native subti. berg and DY. (o--o) PMS-subtilisin (o--o) P>!S-subtillsin DY, and (A--r)native sublisin Carlsberg; tilisin DY.

At

lower

observed values

pHs

and of

of

cence

changes

phan.

Khen

7)

the

quenched

pHs

of

changes

300

from

nm

the

t1ve

enzymes

pclar

and

that

in acidic

subtilisin

2 shotvs the

pH-dependence

(QTyr ) Of

275

nm)

and

the

tyrosyl

quenched

the

due

to

the

of

of

enzlnes

but

subtrlisins

of

atPEI

after

ti-e lndolegroup

at

same

is valid

tnat

relative

ratio

of

that

of

the

berg

is

0.86.

the

degree

the

case

respectrve

for shown

case

the

for

tyrosine

tyrosine

the

total

the

was

emission

(~~~=300

of

in DY

PH

nn)_

calcu-

AtpH

group(s)

of

DY

'esposed' that

the

tne

tyrosyl Tne

the

Carlsberg

are

less

protease.

tyrosyl

belo'ir

q%ith a quenchp H-

(1-e.

residues

and

of

P;lS-suotillsinCarls-

is

0.35

(ref.1)

which

at the surface of the ;Mcrovalue

of

residues

increase

(more

enzyme

'esposed'

=

lS

fluorescenceRTflr of

7)

RMS-subtilisin residues

na-

(he>

Carlsberg

ionogenic

tyrosrne at

PHs

fluorescence

Carlsberg.QTvr

emission

residues.

protein

groups

of

(ref.6)

resposure'

'buried'

in the R Tyr groups in thrs

of

for

value of

of

acid

RTqr

It was

yreld

fluorescence

amino

is a typical molecule.

quantum the

P:S-

acidic

=5.2. Conformational changes could not influence tne aPP the inhibited enzyme is stableintne ing till pH 5.0 oecause region 5.0-10.0 (Fig. 1). The

lobsexci-

?K

the

its extent

observed

concluded

PMS-subtilisin

protonatron

higher

stable

contribution

fluorescence

micro-

PMS-derlvatlves.

between

tryptophan

the

conditions

more

PXS-subtilisin

uslngdifferencespectra

6.4

The

ataci-

were

solutions

their

tyrosyl

changes

Itcouldbe

DY.

with

the

curves

alter

to a much

two

near

strongly by

these

native

the

is to someextent

in comparison

yield

the

hydrophobrc.

is

wnlch

Similar

on

(at pH

three-dimensional

Under

contributes

of

trypto-

single

titration

DY

cha-

the

ring

the

and

tryptophan.

Studies

to

in

Carlsberg

showed

inhibited

r1g. quantum

changes

the

are

fluores-

is determined in

was

reached

which

state

indole

emission

spectra 1).

of

AX

H--induced

native

unique

fluorescence.

Carlsberg

than

the

single

fluorescence

subtllrsin

the

their

transitions

quenched

(Frg.

(ref.4).

position

and

may. respectively,

nm,

in

maximum h

'dequenching'

are of

protein

4.5 at

to a

subtlllsins

not

emission wider:

emission

and

the

the

48-49

considerable

of 1s

than

lated

due

observed

total

tation

and

proteases

reflect

the

ill

are

The

emission

er

nm

fluorescence

structures

the

became

tryptophan

the

environment

to

of

width

(refs.l,S)

residues. die

shift

band

315-317

racterlstlc

to

a red

the

showed to

the

RTLr

depends

on

hrgner

in

being than rhat solvent

3 times) phenolic than

the

258

0.14

0.10

0.06

ox)2 1

I

8

I

I

2

4

6

8

IO PH

of Fig. 2. Effect quantum yields of Ac-Tyr-NH2 (x--x) tained after 24 h The

quantum

subtrlisin shown

Probably

the from

nearby

groups.

the

2.

of

results

tryptophan

fluorescence

is dependent

The

a protonatlon

changes

DY

yield

Carlsberg

In Fig.

with

pH on the tyrosine (e---0) and tryptophan (O--o) subtliisin Carlsberg andon the model compounds and Ac-Trp-NH2 (r--r). The reported data were obincubation at each pH.

effect

of

on

mentioned

functional

fluorescence

6.0

due

3.5

Between

obtalned

pH under

to

6-11 the

Trp same

could

be

of

PMS-

pH

6 as

1s

associated

or conformationalchanges.

enhancementwhen

a decrease Q

(QTrpf below

acidity

above

groups

Hf-Induced to

the

in

the

is constant, condrtions

the

pH

quenching

by

in contrast with

to

subtilisin

(ref.7).

REFERENCES N.

Genov, M. Shopova, R. Boteva, G. Jori and F. Ricchelli, Biochem. J., 207 (1982) 193-200. R.B. Homer and S.R. Allsopp, Blochim. Brophys. Acta, 434 (1976) 297-310. S.S. Lehrer, Biochemistry 10 (1971) 3254-3263. and M.N. Ivkova, Photochem. PhoE.A. Bursteln, N.S. Vedenkina tobiol. 78 (1973) 263-279. S. Omar, R.A. Raubach and T. Schlelch, BiochemlstM.F. Brown, ry 16 (1977) 987-992. F. Quadrifoglro, R.W. Codgill and C. Crane-RoV. Giancotti, binson, Biochrm. Biophys. Acta 624 (1980) 60-65. G. Jori, B. Filippi, R. Boteva, bl. Shopova and F. Ricchelii, Biochem. J. 207 (1982) 201-205. N. Genov.