Chemical modifications of amino groups of trypsin

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 36, No. 6, 1969

CHEMICAL

MODIFICATIONS A.

OF AMINO

Nureddin

and

T.

Department of Biochemistry, School of Medicine, Nashville,

Received

GROUPS

OF TRYPSIN”

Inagami

Vanderbilt University Tennessee 37203

July 29, 1969 SUMMARY

Reactivity of amino groups of chromatographically homogenious (3-trypsin was studied by chemical modification. Guanidination by 1 -amidinyl-3, 5-dimethyl pyrazole nitrate led to modification of 10 out of 14 lysyl residues per molecule of trypsin. The guanidinating reagent was found to be a competitive inhibitor of trypsin. Extent of acetamidination by methyl acetimidate hydrochloride was affected by pH, temperature and the presence of various competitive inhibitors. All 14 c-amino groups of lysyl residues could be acetamidinated in the presence of the competitive inhibitors at pH 9. 5 and 25” without loss of enzyme activity. When 11 or more lysyl residues are modified by acetamidination, the enzyme is stable against autolysis in the absence of Caft. The enzyme with 10 of its 14 lysyl residues guanidinated loses its enzyme activity by autolysis at neutral pH at a rate comparable with unmodified enzyme. Studies very

rapid

of trypsin

not susceptible guanidinated digestion guanidination

* **

cause

and 2 arginyl

at neutral

Splitting

autolysis.

to be the primary lysyl

molecule

of lysyl

residues

(1).

or amidinated As the first with

ADMP””

Modification

peptide

to stabilize bond

and amidination

by NIH grant 1-amidinyl-3, hydrochloride.

999

easy

due to a

bonds

is thought

molecule

of these

contains

groups

the enzyme.

to stabilize

with

MA1 were

GM-14703. 5-dimethyl

14

to a form

is not susceptible

step of an attempt

This work was supported Abbreviations are: ADMP, MAI, methyl acetimidate

peptide

A trypsin

is expected

lysyl

not been

and arginyl

of the autolysis.

to the autolysis

(2,3).

pH have

A

to tryptic

trypsin, studied.

pyrazole

nitrate;

BIOCHEMICAL

Vol. 36, No. 6, 1969

Acylation

was

in trypsin

(4-6).

acetylation

does

E -amino

and

are

it

is

site.

not is

the

not

likely

affect

ination

and

guanidination

hence

it

of the

amino

groups

and

the

is

amino

the

causes

by eliminating, Amination

charge

of such

at neutral

pH,

and

groups on the

modification

in the

9) and,

enzyme.

This

on trypsin

and

reactivity

specific

to amino state

other

modification,

effect

amid-

charge hand,

are unaltered

describes

on the

of

of the

leave

communication its

for reagents,

positive

on the

active

conformational

physical-chemical the

upon

the

chemical

is not

guanidination,

(3,

near

acylating

acylation

change

( -amino

Reagents

in the

the

in trypsin,

subsequent

than

changes

Furthermore,

the

group

groups.

reactive

subtle

activity,

be located

and

of these less

a drastic

may

groups

a complete

However, functional

binding

reactivity

to detect

to amino

net

substrate

groups.

group.

specific

of them

some

are

expected

protein

that

that

of enzyme

catalysis.

occurring

the

can

for

of amino

observed

loss

frequently

case,

importance

(2) has

a significant

essential

is the

change

the

Labouesse

cause

most

very

If it

to determine

Since

group

groups

used

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

application

stability

of the

enzyme.

MATERIALS S-Trypsin, according

to the

experiments. Bannard MAI-1-14C methanol The

obtained

et al. was

(8),

and

with

METHOD

chromatography

of Schroeder

was

prepared

as described reaction

by the

procedure

ADMP

AND

prepared was

Shaw

according

recrystallized

from

from

was

and

carried

1000

used

method

ethanol and

Ludwig

(Worthington)

(7) was

to the

acetonitril-l-14C

by Hunter ADMP

and

of trypsin

(m.p.,

in

of 167-168”).

anhydrous

(3). out

essentially

all

as described

Vol. 36, No. 6, 1969

(9).

by Habeeb of amidination counted

Details with

gel

Protein

(Packard).

E:g”

out

phenylated

6 N HCl

at

Spa&man

110’.

over

mated

legend

scintillation calibrated

1.

counter with

1.

The

using

internal

hydrolyzed

with

and

Singer

in evacuated analysis

was

method

Radioactivity

was

a thixo-

standards.

spectrophotometrically

to Wofsy

acid

to Fig.

in Table

Dinitrophenylation

Amino

et al.

Modification out

(10).

was

given

determined

according

protein

in the

is

It was

= 14.4

carried

-l*C

was

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

given

Tri-Carb

concentration

using

are

MAI-

in a Packard

tropic

was

BIOCHEMICAL

at

280 mp

fluorodinitrobenzene

(12).

The

sealed

tubes

made

dinitro-

according

with to

(13).

RESULTS

AND

ADMP:

The

with

a period

of 205 hours.

by determining

the

content

DISCUSSION modification The

extent

of lysine

reaction

was

carried

of reaction

was

esti-

and

homoarginine.

Fig.

HOMOARGININE_

TIME, HOUR Fig.

1:

Modification 1 -amidinyl-3,5

of

e-amino -dimethyl

groups pyrazole.

of fl-trypsin

The reaction was carried out with 0.3 M ADMP, 0.05 M CaC12, and Aliquots taken at pH 9.5 and 0’. with 2 M sodium acetate buffer pH 0.7 mM HCl for 3 days at 4O and o) formation of homoarginine, to---lysine remaining.

1001

with

a mixture containing 0.1% p-trypsin at intervals were acidified 3. 5, dialyzed against lyophilized. (o -0)

1

Vol. 36, No. 6, 1969

shows in

that

10 lysyl and

120 hours,

a slow

ensuing

inhibitor, of the

these

residues

1.

(14),

presence

Amidination

of P-trypsin

with

guanidinated

in 205

or

of a competitive

others. reagent

are

is modified absence not

3 to 4 lysyl

than

modifying

of trypsin

Ca ++ does

and

Thus

to ADMP to the

molecule residue

reaction.

reactivity residues

per

The

reaction.

modification

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

an additional

benzamidine

different

Table

BIOCHEMICAL

The can

affect

rate

residues lack

be due

methyl

the

hours

by

and

show

a

of reactivity to their

acetimidate

extent

of

location - 1 - 14C

To a 10 ml reaction mixture containing 0. 1% trypsin was The added 22 mg of methyl acetimidate every 20 minutes. pH was adjusted first manually with 5 N NaOH, then it was maintained at a desired value with 1 N HCl using a Radiometer pH-stat assembly.

Moles of acetamidinylmoles of lysyl residues per mole of trypsin

14C incorporated and remaining (in parenthes)

Competitive PH

Temp. (“Cl

Reaction period (hour)

25

2 l/2

None

1

Benzamidinea

14.2 14.0

inhibitor

Butylguanidineb

ADMPZ

(o)*

14. 5

14.2

9.5 5

8.5

2

13.2

1

14.0

14.2

25

1

14.2

13.7

5

2

13.2

13.0

1

11.2

* Moles of lysyl residues as c -dinitrophenyl-L-lysine 2 0.50 M, k 0.25 M,

(0.5)*

13. 8

13.2

14.0 (2.0)”

12.5

(3.1)”

11.7

remaining per by amino c 0.30 M 1002

mole acid

of enzyme analyzer

determined

BIOCHEMICAL

Vol. 36, No. 6, 1969

inside

the

enzyme.

competitive that

inhibitor

the

bound

However,

4 lysyl

at the

residues

active

also

trypsin. residues inhibitors at pH modified can

be

butyl

pH

and

pH

8.5

and

(14),

presence

the

complete

the

amidination

J. H. Reynolds MA1 according

the same

is

the

12 lysyl

the

residues

are

11 residues

inhibitor,

terminal This

which results

of time.

to the

Fig.

retains

has

stability effect

13 lysyl residues communication.

to do.

2 trypsin only

at room

trypsin The

in

that

fails

stabilizes

p-trypsin

*

indicates

shown

residues

3

isoleucyl

benzamidine

of incubation

1003

modification

and

amino

condition.

of Ca ++,

comparable

competitive

respectively.

acetamidinated

has modified to his personal

that

of protein

The

period

14 lysyl

mole

of 14 lysyl

fully

all

of 2 and

residue

24 hours

of

appearance

lysyl

after

against

than

by the

this

absence

reactive

to modify

competitive

that

trypsin:

In the activity

after

likely ADMP

inhibitor

of benzamidine,

under

modification

8. 1, whereas

activity

by the

to note

of another

one

of modified

autolysis.

its

to be a more

more

competitive

only

per

unmodified

original

found seems

by far

effect

indicates

protects

is

is possible

as evidenced

chromatography

the

reagent

however,

C-dinitrophenyl-L-lysine

that

the

a strong

5”,

presence

indicate

it

protective

in the

Stability

of its

their

reacted

guanidine

mM,

1.4

It is interesting

in the

remains

against

25”.

2 hours

Thin-layer

Ki

MAI

1, it

after

of

residue

is not

and

exert

1 -n- butylguanidine moles

been

from

-14C:

in Table

9.5

At

9.5.

has

shielded

MAI-

reagent

shown

do not

ADMP

site.

this

at pH

with

are

with

As

since

of trypsin

Modification ADMP,,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

197%

temperature still

90%

afforded

of Cat+

at pH

by

on

8.5

of

by

Vol. 36, No. 6, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o-0 Fig.

2:

Stability

of modified

IO 20 30 TIME, HOUR

40

and

P-trypsin.

unmodified

The loss of enzyme activity during incubation of a 0.04% solution of P-trypsin in 0.2 M Tris-HCl buffer pH 8.06 at 25’ was followed by determining the activity of aliquots taken at intervals by a Radiometer pH-stat assembly using benzoyl-L-arginine ethyl ester as substrate (11). t) trypsin with all 14 lysyl residues acetamidinated, --00) unmodified trypsin incubated with 0. 05 M CaC12, trypsin with 11 lysyl residues acetamidinated by k -& a reaction at pH 8.5, 5” for 2 hours in the presence of butylguanidine, (0-0) trypsin with 10 lysyl residues guanidinated to homoarginine, (o -0) unmodified trypsin incubated in the absence of Cat+.

t+-

unmodified

p-trypsin.

of 10 lysyl

residues

trypsin lysyl

towards

bond

does

stabilizes does.

in trypsin

which

active

site.

in our

laboratory.

Identification

is

to note

significantly

On the it

Thus

interesting

not

autolysis.

residues

modification

It is

to the

there essential of this

change

other

hand,

same

seems for

to be

the the

the

extent

one

bond

modification

stability

of

modification

as the

maintaining

particular

1004

that

complete

critical the is

of 11

lysyl integrity

under

peptide of the

investigation

Vol. 36, No. 6, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENT

Miss Mrs.

The authors L. H. Clack Alice Knapp

express their thanks to Mr. for performing amino acid for skillful assistance.

J. D. analyses

Ford and

and to

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Walsh, K. A. and Neurath, H., Proc. Nat. Acad. Sci. U.S.A., 52, 884 (1964). Shields, G. S., Hill, R. L. and Smith, E. L., J. Biol. Chem. t 234, 1749 (1957). Hunter, M. J., and Ludwig, M. L., J. Amer. Chem. Sot., 84, 3491 (1962). ‘Gbouesse, J. and Gervais, M., Eur. J. Biochem. 2, 214 (1967). Terminiello, L., Sriram, J., Bier, M. and Nord, F. F., Arch. Biochem. Biophys., 57, 252 (1955). Westman, T. L., Biochem. Biophys. R. C. 35, 313 (1969). Schroeder, D. D. and Shaw, E., J. Biol. Chem., 243, 2943 (1968). Bannard, R. A. B., Casselman, A. A., Cockburn, W. F. and Brown, G. M., Can. J. Chem. 36, 1541 (1958). Habeeb, A. F. S. A. , Can. J. Biochem. Physiol., 38, 493 (1960). Davie, E. W. and Neurath, H., J. Biol. Chem., 212, 515 (1955). Tietze, F., J. Biol. Chem., 204, 1 (1953). Wofsy, L. and Singer, S. J., Biochemistry, 2, 104 (1963). Spa&man, D. H., Stein, W. H. and Moore, S., Anal. Chem. 30, 1190 (1958). Mares-Guia, M. and Shaw, E., J. Biol. Chem., 240, 1579 (1965). Inagami, T. and York, S. S., Biochemistry, 1, 4G (1968).

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