Vol. 68, No. 4, 1976

BIOCHEMICAL

EVIDENCE FOR ONE ESSENTIAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TRYPTOPHAN RESIDUE AT THE ACTIVE

C. Schwabe and Department of Medical University Charleston, Received

December

SITE OF RELAXIN.

S. A. Braddon Biochemistry of South Carolina S.C. 29401

9,1975

SUMMARY: The hormone relaxin, which is responsible for the rapid widening of the birth channel in mammals prior to parturition, was purified from hog ovarian extracts and shown to be homogeneous by exclusion chromatography in 6 M guanidine hydrochloride and SDS gel electrophoresis. Of the two disulfide-linked chains that comprise relaxin, the larger chain was shown to contain two tryptophan residues, one of which could be completely oxidized in native relaxin without measurable effect on its biological activity. Oxidation of the second residue completely inactivated the hormone. Modifications of lysine side chains or carboxymethylation of a single methionine residue at low pH did not impair the effectiveness of relaxin. Ovarian capable

mice activity

pigs

(2)

of Health

molecular

weight

disulfide

bonds

because

selectively

Evidence

is

with

presented

tryptophan

residues

side

is

chain

molecule

mouse bioassay

contains

did

not

that

relaxin Extensive

essential. lead

form from

hormone

of the

is

residues

without

causing

modification activity

modifications

to any significant

linked quite

be removed

which chain

the

other

of other diminution

by clear

after

that

the

larger

can be oxicleavage.

of one of the

while

National

to have a

are not

however,

for

pregnant

the

two chains

can only clear,

fresh

from

appears

structure

that It

responsible

available

two tryptophan

paper

eliminates

not

(3).

N-bromosuccinimide in this

extract

factor

in estrogen-primed

hormone

purified

peptides

of relaxin

chains

a nonsteroid

1) , and to contain

details

interacting

peptide

ovarian

daltons

The finer

(2).

two relaxin

in highly

contain symphysis

the

acid-acetone

of 5600-5800

and alkylation

of the

Relaxin,

The porcine

(3).

of tenaciously

reduction

(1).

or an ovarian

of species

in the pubic

can now be prepared

Institutes

relaxin

remodeling

or guinea

hog ovaries

dized

from a variety

of stimulating

female this

extracts

two

tryptophan parts

of potency

of the in the

(3).

')This result from our own work is consistent with the relaxin composition published by Sherwood and O'Byrne. They, however, value of 6400 daltons obtained by analytical centrifugation. 1126

Copyright 0 I976 by Academic Press, Inc. All rights of reproduction in any form resert,ed.

amino prefer

acid the

BIOCHEMICAL

Vol. 68, No. 4, 1976

MATERIALS:

Relaxin

through

the National

addition

a purified

of Dr.

B. Steinetz

parations

were

bioassay

system.

cals

was purified Institute sample (Ciba

Chemical

--et al.(4).

The animals

Laboratories

ligament were

in 0.1 ml sesame oil

containing

pg of a relaxin

later

and the

interpubic

at the

narrowest

illumination

N-Bromosuccinimide dissolved

oxidation:

in exactly

obtained for

from

light

to the

2 ul-aliquots

of a 10 mM solution

when the

The difference relaxin

absorption

was obtained

and Witkop

bromide

from the

were

by utilizing

female

mice,

obtained

the

distance

from

5 ug of estradiol

After

sample

22 hours

buffer

(0.2

a sample

spectrum for

accomplished

1127

trans-

Samples

for 25,

the

mg) was

M, pH 4.7'). (380-250

between

bioassay

and evaluated

(5).

under

18

Using nm) was

at 280 nm was corrected 380 and 340 nm

was removed.

by adding

at 280 rsn between

4-B

sacrificed

(1.5

spectrum

of relaxin

of the

each

caliper.

of relaxin

an initial

of NBS.

were

was measured

a millimeter

direct

by Steinetz

purchased

with

injection.

acid-NaOH

recording

(CNBr)

(NBS),

purification.

at 280 nm had decreased

in absorbance

Chemi-

N-Bromosuccinimide

The animals

with

time

Organic

in mice as described

The absorption

was then

Eastman

of 0.1 ml of 1% benzopurpurine

ligament

by extrapolation

of tryptophan

Co.

further

preparation.

At this

from

and cyanogen

A salt-free

solution.

280 run region.

in the

by subcutaneous

spectrophotometer,

scattering

oxidation

drawn

this

effectiveness

Each mouse was primed

1 ml of acetic

a Cary 15 recording

equal

was accomplished

point

courtesy Both pre-

Chemical

injection

the

In

New York).

g ICR virgin

Va. ).

obtained

Ardsley,

formation

16-20

a subcutaneous

hours

(TNBS),

of relaxin

mouse received

through

4-B was obtained Sigma

extract

and Human Development.

and possessed

acid

(Dublin,

ovarian

was obtained

Corporation,

from

The bioassay

l-3

Health

Co. and used without

of interpubic

benzoate

of Child

equal

benzoate

measurement

Flow

Geigy

sulfonic

EXPERIMENTS:

an enriched

Benzopurpurine

trinitrobenzene from Pierce

from

of relaxin

chemically

and estradiol

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The

to the

photometer

bioassays

were

cell

with-

50, 80, and 100 percent.

titrated

and untitrated

as described

by Spande

Vol. 68, No. 4, 1976

BIOCHEMICAL

Trinitrobenzene

sulfonic

was performed

essentially

in 1 ml sodium TNHS in water sorption

borate were

plateau

During

the

in small

acid

portions

according buffer

the

redissolved

indicating

the

withdrawn

of methionine.

in an appropriate

of the

precipitating

lysine

relaxin

At time

for

acid

material

of benzopurpurine

an ab-

in

were

added

solution.

performed.

in 1 ml of 70% formic

bromide. amino

of

modification.

crystals

was similarly

The remaining amount

of a 10 mM solution at 367 nm until

(1 mg) was stirred

10 mg of cyanogen

by TNBS

(1 mg) was dissolved

hydrochloride

chloride

Relaxin

25 ~1 each were

and 10 ul

completion

dansyl

of lysine

Relaxin

was monitored

slowly

0.1 N HCl with

disappearance

The reaction

with

(6).

M, pH 8.3)

to maintain

treatment:

48, and 72 hr,

to Stark

guanidine

Cyanogen or

The modification

of the reaction

of lysine

acid

(0.1

added.

The modification bromide

modification:

was reached

course

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

intervals

analysis

of 0, 24, to demonstrate

was lyophilized 4-B for

and

bioassay.

0.8

0.6

240

260

280

300

320

340

360

x (nm)

Fig.

1

The stepwise oxidation of tryptophan in purified relaxin. N-Bromosuccinimide in water was added directly to the spectrophotometer cells prior to each scan depicted. See text for details. The decrease of absorbance at 280 nm as a function of NBS concentration can be evaluated without complication because of the lack of tryrosine in relaxin. The upper line represents the unmodified relaxin spectrum; the lowest line (at 280 nm) depicts the spectrum of the completely oxidized relaxin.

1128

BIOCHEMICAL

Vol. 68, No. 4, 1976

Methionine

The alkylation

modification:

guanidine

hydrochloride

(1 mg) was allowed

buffered to react

16 hr at room temperature 0.9 x 30 cm column Incorporation

of Sephadex

indicated

80-90s

A chain

of relaxin

2) .

The reaction

as clearly

shown in Figure

that

does not

second

equivalent

dation.

Under

chain

at the

clusion the

should

absorbing

the effect indole

conditions

side

did

not occur.

If

of tryptophan

scission

later

from

In contrast,

chains.

oxidation

had occurred, the if

column scission

have remained The result activity

to an oxindole

the

residue

it per relaxin

to its

of oxi-

degree

of the relaxin

was demonstrated

after

by ex-

reduction,

utilizing

has an absorption

oxindole-containing

the remaining

in Figure

fragment

intact the

elution

concomitant

second species

volume

3 clearly

is due to the

without

By

of the

scission

at the normal

on relaxin

2.

indicates

conversion chain

for

the that

of the cleavage.

2) By analogy with insulin the smaller chain (22 residues) is nomenclature, and the larger chain containing the aromatic amino designated the A chain, acids is designed the B chain (30 residues).

1129

of

disappearance

had not occurred,

depicted

amounts

of oxidation

(oxindole)

the

than

small

The results

This

relaxin

and progressive

of tryptophan

experiments

in the

molecule.

proportional

of the NBS-treated

of biological chain

of these

residue

to determine

levels

whereas

is roughly

residue

at 250 nm should relaxin

different

on a

hydrolyzed

shown in Figure

activity

the product

of relaxin.

are

for

was possible

relaxin

inhibit

tryptophan

have eluted

in the

of very

it

of one equivalent

of tryptophan

250 nm.

scanning

of oxidation

hormonal

of the methionine

the addition

at several

desalted

NBS was instantaneous

the modification

the

that

band near

reduced

relaxin

chromatography

fact

chain

degrees

acid

M ammonium bicarbonate.

analysis single

6M

in

The relaxin

of 3 H iodoacetic

in 0.05

acid

with

Through

1.

(7).

and subsequently

of the

of tryptophan

at varying

became clear

and amino

was performed (pH 4.5)

weight

Superfine

(2 ~1) and repetitive

the modified

molecule

G-25

of tryptophan

of oxidation

of bioassays

an equal

modification

RESULTS:

assaying

with

0.1 M acetate

in 1 ml of buffer

peptide

the degree

of methionine

with

of radioactivity

the NBS solution

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 68, No. 4, 1976

I

BIOCHEMICAL

2

3

I

2

AND BIOPHYSICAL

3

I

2

RELAXIN 0

2

residue

scission

by cyanogen

of relaxin. N-terminal residue

2

3

I

2

3

83

40

100

OXIDATION

This

is

residue

The reaction residues

Injection

of the

purpurine

4-B,

is not

an important

These

leads

particularly of the A chain with

of relaxin suspension showed

factor. water results

interesting

since

of relaxin. acid

relaxin

of this

single

of the

hormonal

is

near

not

hormonal

diminish

derivative,

tyrosylated

and shown to have I.

acid

the

activity.

product

(1.8

of

analysis).

emulsified indicating

a heavily

1130

activity

of the methionine

to an insoluble

in Table

hormone

Modification

activity

was tested

methionine

the methionine

as shown by amino

relaxin

are summarized

did

leads

In addition,

insoluble

at the

abolition

were modified

unimpaired

of relaxin

to complete

iodoacetic

of TNBS with

3 lysine

was totally

of the A chain

bromide

by alkylation

activity.

I

The activity of relaxin in the mouse bioassay after varying degrees of tryptophan oxidation by NBS. At 0, 12, 40, 83, and 100% oxidation as determined spectrophotometrically aliquots of the relaxin solution were withdrawn for bioassay. At each level of oxidation three groups of mice were injected with the treated relaxin (i.e. 1, 2 or 3 lig/ mouse). The shaded bar represents the mean untreated control value + S.E.M.; 0, 1 pg relaxin, mean value for n = 10; 0, 2 ug relaxin, mean value for n = 10; +, 3 ug relaxin, mean value for n = 10.

By contrast,

that

3

(pg)

12 PERCENT

Fig.

RESEARCH COMMUNICATIONS

with

that

solubility

relaxin full

benzo-

relaxin

sample

vol.

68,

BIOCHEMICAL

4, 1976

NO.

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

,/,--.--o 0

4

5

6

ELUTION

Fig.

3

Chromatography hydrochloride

7

9

TIME

9

IO

II

(hours)

of reduced relaxin after NBS oxidation in 6 M guanidine on a 0.9 x 30 cm column of Sephadex G-50 Superfine.

0-0

Absorbance

due to peptide

G-G

Absorbance

due to oxidation

bonds

of reduced

product

relaxin.

of tryptophan.

TABLE I BIOASSAY OF RELAXIN AND SEVERAL MODIFIED RELAXIN MOLECULES. Study Treatment Mean P.S.L.

BP* mm

S.E.M. n * +

1

Benzopurpurine

Study Relaxin

TNBS+

BP*

Relaxin

2 Tyrosyl-

CNBr++

0.29

1.32

1.45

0.42

1.54

1.26

0.48

0.08

0.24

0.15

0.03

0.20

o .16

0.04

15

15

15

15

15

15

15

4-B

TNBS = Trinitrobenzenesulfonate

P.S.L.

= Pubic

S.E.M.

= Standard

symphysis error

length of mean

++

CNBr = Cyanogen bromide n = Number of mice. Several chemical modifications of the relaxin molecule were studied in terms of the effect on the bioassay on mice (see EXPERIMENTS). The data are given as the means of the measured pubic symphysis ligaments in millimeters (P.S.L. errors of the means (S.E.M.) and the number mm). Also given are the standard of mice/groups (n). A 1% benzopurpurin 4-B (BP) solution was used as a carrier vehicle for the various relaxin samples as well as a control for the bioassay. Study 1: relaxin (2 rig/mouse), trinitrobenzenesulfonate-treated relaxin (TNBS, 3 Ug/mouse); Study 2: relaxin (1 rig/mouse), tyroxyl-relaxin (tyrosyl-, 2 ng/ mouse), and cyanogen bromide treated relaxin (CNBr, 2 ug/mouse); see EXPERIMENTS for the modification procedures.

DISCUSSION: Extensive chains

The results modification

with

TNBS,

obtained of relaxin

(b ') conversion

in this as achieved of positive

1131

study

were

by (a) lysine

rather

surprising.

reaction side

chains

of lysine

side

to negative

Vol. 68, No. 4, 1976

ones by succinylation, not

influence

to the

as the earlier is

(essential)

oxidized

while

of large

of relaxin.

of relaxin also

was the

fact

that

by NBS.

This

that

natural

the

result,

results

chains lend

of a residue

faster

to the conclusion to the

tryptophan

is

than that

surrounding

in turn,

did

a greater

one tryptophan

sufficient>7

leading

not as accessible

side

by the oxidation

at a rate

residue,

tyrosine These

activity

preferentially

is

RESEARCH COMMUNICATIONS

the

solvent

consistent

fluorescence

with in relaxin

(3). of the

a tightly

two tryptophan effect

showing

quenched

On the basis

out

residue

one readily

severely

activity

tryptophan

tryptophan

is

(c) the

Surprising

experiments

relaxin

addition

be oxidized

second

essential

and

elimination

residue.

can apparently the

AND BIOPHYSICAL

the biological

significance tryptophan

BIOCHEMICAL

coiled

residues. the

second

above

results

two chain

it

seems reasonable

molecule

One of the

tryptophan

residue

is essential

and that residues for

to conclude

its

B chain

contains

can be modified

the hormonal

that

with-

function.

REFERENCES Hisaw, F. L., (1926) Proc. Sot. Exptl. Biol. Meet. 2, 551-66h. Sherwood, D. C., and O'Byrne,E. (1974) Arch. Biochem. Biophys. l&I, 185-196. Schwabe, C. Unpublished observation. 43: Steinetz, B. G., Beach, V. L., Kroc, R. L., Stasilli, N. R., Nussbaum, R. E ., Nemith, P. J., and Dun, R. K. (1960) Endocrinol. 67, 102-114. B. (1967) in Methods in Enzymology (C. H. W. 5. Spande, T. F., and Witkop, Hirs, Ed.) Academic Press, N. Y., 2, pp. 498-522. 6. Stark, G. R. (1970) in Advances in Protein Chemistry (C. B. Anfinsen, Jr., J. T. Edsall, and F. M. Richards, Eds.) Academic Press, N. Y., 4, pp. 261308. B., Degani, Y., and Patchornik, A. (1970) in 7. Spande, T. F., Witkop, Advances in Protein Chemistry (C. B. Anfinsen, Jr., J. T. Edsall, and F. M. Richards, Eds.) Academic Press, N. Y., 4, pp. 97-260.

1. 2.

1132