Denaturation of proteins in 8M urea as monitored by tryptophan fluorescence: Trypsin, trypsinogen and some derivatives

BIOCHEMICAL

Vol. 30, No. 5, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DENATURATION OF PROTEINS IN 8& UREA AS MONITORED BY TRYPTOPHAN FLUORESCENCE: TRYPSIN,

TRYPSINOGEN AND SOME DERIVATIVES"

T. R. Hopkins** and John D. Spikes Department of Molecular and Genetic Biology University of Utah Salt Lake City, Utah 84112

Received

February

Tryptophan

5, 1968

residues

in proteins

maximum at approximately light. the

Since

the

emission

residues

a sensitive

monitor

al,

We previously

1964).

chymotrypsinogen dependent the

turation the

is

as well

when the

altered,

immediate

protein

which

examined

in this

rate

constant

is

in the

around

the

(Steiner

is

a time-

by measurement The rate

1967). first-order

of the

&

of chymotrypsin,

in 8 4 urea

apparent

a measure

of

protein

followed

and Spikes,

way follows

location

has been used as

derivatives

can be conveniently (Hopkins

as the

the denaturation

and some chymotrypsin

a

290 nm ultraviolet

fluorescence

that

band with

environment

changes

reported

fluorescence

calculated

of fluorescence

of conformational

reaction

protein

with

is changed

fluorescing

in a broad

34C nm when excited

intensity

peak

fluoresce

of dena-

kinetics

stability

of

and

of the protein

to 8 4 urea. The present (TG),

paper

describes

diisopropyl-fluorophosphate

* This research was supported No. AT(ll-1) 875. $L ?C Present address: Chemistry, University

similar

studies

treated

trypsin

by U.S.Atomic

Department of Chemistry, of Minnesota, Minneapolis,

540

using

trypsin,

(DIP-T)

Energy

and l-chloro-2-

Commission

Laboratory Minnesota

trypsinogen

under

of Biophysical 554.55.

Contract

Vol. 30, No. 5, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tosylamido-7-amino-2-heptanone inactive

fo.rm of trypsin

phosphate; site

treated

the

region

residue

inhibitor

of the

derivative

produced

enzyme

of trypsin located

reacts

in the

(Dixon

active

(TLCK-T).

by treatment with

in which

trypsin

a serine

et al., the

is

an

diisopropylfluoro-

residue

located

TLCK-T

1958).

reagent

site

MINUTES

with

DIP-T

has reacted

in the active

is

an inactive

with

a histidine

(Shaw -et -m al f 1965).

IN 811 UREA

Fiqure 1. The time-course of the fluorescence transitions of trypsin(T), trypsinogen (TG), and DIP-trypsin (DIP-T) in 8 fi urea at 25.6oC. The solutions contained 0.5 mg/ml protein and were buffered with 0.25 fi sodium phosphate, pH 7.2. The fluorescence was measured at 356 nm using 290 nm exciting light.

Like

the chymotrypsin

displayed

a red-shift in

fluorescent

No shift

in fluorescence

The rate

of the

was increased

DIP-T,

all

from

apparent

shift

when the

contains

in 5 &l urea

6 4 to 8 &

solutions

the 1.

constants

541

for

concentra-

of trypsin,

of appearance

The denaturation (k)

and 30%.

as the urea

denaturation rate

8 & urea.

at pH 7.2

rapidly

The urea

by measuring

and rate

solution

increased

peak as shown in Fig. first-order

in buffered

peak from 341 nm to 356 nm and an

was observed

fluorescence

and TG was followed

356 nm emission

intensity

above proteins

the

in the emission

increase

tion

family,

of the

reactions

the denaturation

were of

BIOCHEMICAL

Vol. 30, No. 5, 1968

trypsin,

TG, DIP-T,

and TLCK-T

seen in Table

I ,TG and DIP-T

These

are

results

the chymotrypsin more rapidly (tiopkins

in

sharp

family than

the

in 8 fi urea

contrast

with

in 8 E urea

studies

where

0.25

differential

in 7.4 x urea

at 20%

ences

relative

difficult

TLCK-T

4.5

3.1

>lOO

temperature

with our

higher theirs

rate

of the

because

It

of differtrypsin and the

agreement.

in 8 ~x-Jurea enzyme with

ours.

for

as theirs,

in excellent

to denaturation

and DIP-T than

constants

of magnitude are

and Laz-

of trypsin

slightly

but

to DIP-T

28oC

at 293 nm, Delaage

our data

same order

was placed

increase Fig.

Trypsinogen

was examined

1-chloro-2-tosylamido-

(TLCK) .

When trypsin

(see

the

of inactivation

7-amino-2-heptanone

urea

derivative

DIP-T

pH conditions,

of trypsin

the course

three-fold

is denatured

of Trypsin,

denaturation

concentration,

of trypsin

The stability during

enzyme

E

pH 7.2,

a first-order

to compare

at 20°C are rates

4 phosphate,

under

in pH and urea

and DIP-T

active

of

I

spectrophotometry

observed

somewhat

on the denaturation

Rate Constants

L 1.1

(1967)

is

trypsin.

in 8 fi Urea.

Solvent:

dunski

than

and the diisopropylphosphate

Transition

k(min-1)

Using

the

As may be

1967).

Fluorescence

Some Derivatives

calculated.

more rapidly

Table First-Order

were

are denatured

zymogen

and Spikes,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(2),

in the Curve

in rate

an excess of its

was not affected

more easily

intermediate

denatured

fluorescence

1) even though

L-arginine-ethylester

the

enzymatic

at this

time.

trypsin

542

of TLCK there

derivative

was an immediate

transition activity

in 8 &j with

The identity is

not known.

benzoylof this

and

Vol. 30, No. 5, 1968

With

increasing

lost

and

as

time

the

shown

in

rate Fig.

of

of

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

exposure

TLCK,

the

to

fluorescence

trypsin

activity

transition

in

was

8 M urea

progressively

increased

(2).

I

I

I

I

I

1

I

I

0

I

2

3

MINUTES

I

I

4

IN EM UREA

Fiqure 2. Fluorescence transitions of trypsin in 8 1 urea at 27% before (B) and after increasing time of incubation (l-4) with TLCK reagent. Trypsin (1201&l) in 0.1 4 Tris buffer, pH 7.7 was incubated at 27% with The enzyme activity of aliquots taken during 1 ti- TLCK for up to one hour. the course of reaction was assayed with benzoyl-L-arginine-ethylester. Percent remaining enzyme activities relative to untreated trypsin incubated under similar conditions were (1)) 99 percent; (2), 59 percent; (3), 17 percent; and (4)) 1 percent. Fluorescence intensities were corrected for absorption by TLCK reagent.

Trypsin

was

denaturation

rates

values was

(see very

F

-9.

TG,

resistant

(3)). to

DIP-T

8 &j urea

as

plot

was

linear

the

slope

(see

of

deviated

to

addition,

the

denaturation II).

of

temperature

upward

(AH* from

The

rate

of

were

of

(AH*

but, 3

and

were

the

calculated denaturation The

at

higher

transition theory,

DIP-T

the

with

lower

complicated.

= 16 kcal/mole)

543

to

rate

8 2 urea

kinetics

pH 7,

fluorescence

absolute in

linearity

at

pH changes

Using

Table

26OC

denaturation with

temperature.

a function up

urea

rapidly

In

parameters

and

to

increasing

sensitive

thermodynamic T,

most

higher

16 kcal/mole).

for in

Arrhenius temperatures

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 30, No. 5, 1968

Fiqure 3. The effect of pH on the first-order constant The solutions transition of trypsin in 8 M urea at 28oC. protein and were buffered with 0.25 _M sodium phosphate.

Table Thermodynamics

of Denaturation

the fluorescence contained 0.5 mg/ml

II

of T, TG, and DIP-T

in 8 g urea

AFS(kcal/mole,

AH*(kcal/mole) 28 + 1

T

for

250)

AS*(E.U.)

20.2

+ 0.1

28 _+ 3

TG

23 f 1

19.3

+ 0.1

*13 + 3

DIP-T

16 _+ 1

19.6

_+ 0.1

-12 + 3

Solvent: 34°C; The data

show that than

stabilities

of DIP-T

group the

to the entropies

Jj phosphate,

TG, 210 - 30%;

in 8 E urea

or to altered

0.25

the

local

binding

tation

that

the altered

tion.

This

conclusion

resulting

One might

of activation

could

are denatured

Relative

to trypsin,

be due either from the

conclude

that

rates

must be accepted

with

544

ranges:

T, 22O -

the

at higher

to changes

large favor

in conformation

of the

inhibitor

differences the

are due to changes caution,

rates

the decreased

attachment

TG and DIP-T

of trypsin, denaturation

Temperature

190 - 26°C.

and TLCK-T

enzyme.

and TLCK-T

protein.

DIP-T,

TG, DIP-T

active

pH 7.2.

however,

in

interprein conformabecause

BIOCHEMICAL

Vol. 30, No. 5, 1968

entropy

is

not always

and Biltonen,

urea-denaturation

Both

studies

chymotrypsin

order

amino

derivatives While

trypsin

composition, of these are

-et -fal , 1966;

changes that

similarities,

our data

(Yapel

and TLCK-T

as monitored

the details

conformation

(Lumry

and those

demonstrated

stabilities

two enzymes consistent

may be quite

with

current

Lumry and Eyring,

changes different

during

those

fluorescence.

the

in 8 @ urea

models

in the first-

rates

of

are similar.

in catalytic

Des-

function

and

and diisopropyl-

to be quite

different.

of enzyme mechanism

Koshland,

the catalytic

involved from

Also,

appear

1954;

related

of pH on the

of the zymogens

in 8 1 urea

previous

to 8 a urea

are different. enzymes

our

by tryptophan

stabilities

two proteolytic

the

extend closely

of the effects

rates

in enzyme conformation

the conformational

of another

show maximal

transition

of these

acid

TG, DIP-T

on derivatives

and trypsin

fluorescence

these

in protein

chymotrypsin,

of 7, although

denaturation pite

on trypsin,

enzyme,

pH region

of changes

in press).

The above data

proteolytic

a measure

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1958) process,

in the catalytic

which

include

they

suggest

mechanism

of

of chymotrypsin.

REFERENCES M. Delaage and M. Lazdunski, Biochem.Biophys.Res. Comm. 28, 390(1967). G.H. Dixon, D.L. Kauffman, and H. Neurath, J.Biol.Chem. m, 1373(1958). T.R. Hopkins and J.D.Spikes, Biochem.Biophys. Res. Comm. 28, 480(1967). D.E.Koshland, Jr. , Proc. Natl. Acad.Sci. (U.S.) 4, 98(1958). R. Lumry and H. Eyring, J.Phys.Chem. j8, llO(l954). R. Lumry and R. Biltonen in ‘~Biological Polymersn Vol. 2 (ed. S.Timasheff and G. Fasman) Chapter 1, in press. E.Shaw, M. Mares-Guia end W. Cohen, Biochem. 4, 2219(1965). R.F. Steiner, R.E. Lippoldt, H.Edelhoch and V. Frattali, Biopolymers Symposia 1, 35511965). A. Yapel, R. Lumry and M.H. Han, Fed. Proc. 3, 650(1966).

545