Identification of a new crosslinking amino acid in elastin

Vol.

21,

No.

6, 1965

BIOCHEMICAL

IDENTIFICATION

OF

A NEW

Carl Franzblau,

F.

Department

AND

CROSSLINKING

Marott

Sinex,

of Biochemistry,

November

Recently,

residues

amino

et al. --

acids

for

in a “pre-elastin”

called

desmosine

side-chain

desmosines

revealed

that

the

(Franzblau be represented

described

by

Dowex crude

isolated

Franzblau

50 x 8 by means X4

fraction

The Model

Supported from the

and

University

d escribed

which

evidence

(Partridge

et-- al., are

function

1964;

IN

Rhoda

School

ELASTIN

Lampidis

of Medicine,

in fact

of elastin

1965).

by the

formula,

The

from

acid

et -- al.,

selected

on a sulfonated

electrophoretic

R electrophoresis

by grants from American Heart

mobility cell

at 300

the National Association

this

called

X4,

lysine

substances, with

four

“,’ aJ., respect

also

presents

1964). to the

previously

data

to show

that

BufferJ

RESULTS

of bovine

method

of HCI

ligamenturn

consists

followed

nuchae

of fmctionation

as on

by rechromatogmphy

of the

resin.

of X4 was studied

575

(Thomas

AND

polystyrene-divinylbenzene

Institutes (65-G-129).

from

These

with

communication

of elastin

solutions

volts.

origin

compounds

enrichment acid,

hitherto

%corboxypentanyl)-lysine.

In brief

with

for

of two

their

1964).

chains

PROCEDURES

hydrolysates

of elution

a-l., pyridinium

amino

N s-&amino

(1965).

t

to show

of elastin

present

in elastin

accrued

quaternary

of an additional

eta- al.,

occurrence

Miller

in crcsslinking

fractions presence

the

has now

EXPERIMENTAL X4 was

ACID

Massachusetts

(1963)

isodesmosine,

of peptide

undescribed X4 can

and

substituents

An examination

Faris

COMMUNICATIONS

12, 1965

Partridge

unrecognized

RESEARCH

AMINO

Barbara

Boston

Boston,

Received

BlOPHYSlCAL

using

Whatman

were:

acetic

used

of Health,

AM-07697

#I acid

and

paper

and

- formic

GM-08062,

a Spinco acid

and

Vol. 21, No. 6, 1965

BIOCHEMICAL

(pH 1.9), pyridine-acetic (pH 10.9).

X

acid (pH 3.5 and pH 6,4),

migrated

4

could estimate

as a single

its isoelectric

Reactions

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

point to be between

The copper

dinitrophenylated,

and the copper stripped

DNP derivative vealed

complex

A spectrum

pipecolic

dinitrophenylation

phenylated

of DNP-alanine

In a Technicon Spackman

--et al.

In contrast,

of X

4

one could infer that this derivative

derivative,

and secondary Nuclear

of X4 formed by

that of the fully dinitro-

a copper complex

was formed.

The

by a model system consisting

a column similar

of that of e-DNP-lysine.

reaction,

phenylated

with

after ninhydrin

of the DNP group and the number of leucine

secondary

of X4 re-

amino acids

amino group.

one could conclude

to that described

formed from the copper complex

efficient

DNP-substituted

the derivative

be reproduced

employing

as a skewed peak which

is reminiscent

FDNB, and the new

of N-substituted

at 440 mt.~. This in part was due to the intrinsic

This behavior

with acid and

and one of DNP-sarccsine.

the DNP de rivative

eluted

carbonate,

peak at 355 mp with a broad shoulder

the step in which X4 could

by treatment

of the copper complex

was almost identical

amino acid analyzer

(l958),

of copper)

high absorption group.

omitting

of the fully dinitrophenylated

of two equivalents

stripped

spectrum

with copper

once more with

of DNP derivatives

of X4

FDNB by the method

by treatment

of the DNP derivative

in two stages had a major absorption

X4 formed directly

spectrum

was then treated

acid and proline.

at 385 mp; in fact its absorption

with

from the DNP derivative

at 385 mp characteristic

such as sarcosine,

directly

of X4 was prepared

of this derivative

isolated.

absorption

pH 8.6 and pH 10.9.

First, X4 was dinitrophenylated

An aliquot

one

(FDNB) of X4 and of the copper complex

of Sanger (1945).

then H2S.

(pH 8.9), and sodium carbonate

band in all systems and, from the data obtained,

with fluorodinitrobenzene

were studied as follows.

barbital

contained

reaction

(and then

showed relatively

absorption

by the dinitrophenyl

From the molar extinction

equivalents two primary

Taken with the spectml that X4 e

by

F contained

yielded

co-

in the ninhydrin

a-amino

groups and one

data of the fully dinitroprimary

a-amino

groups

amino groups in a mtio of 2: 1. magnetic

resonance

studies were

576

performed

with X4 in both deuterium

oxide

Vol.

21,

and

No.

BIOCHEMICAL

6, 1965

in trifluoroacetic

absence

in X

(Franzblau

of C-CH3

et-- al.,

of amino group

4

acid.

acids

such

was

spectrum difference.

methods

nature

of X4 precluded

are

of Van

of X

in fact

is shown

Form01 8.0

and

one

titration with

a pK at

59 micro-equivalents Thus,

these At

structure of this

compound

in Figure

chamcteristic

the

at 3.17

and

carbon

primary

Van

and

Slyke

results

a-amino found

obtained

groups

almost due

and

by

an imino

identical

except

e-NH:

group,

to the

of samples,

contents

(1941).

analyses

one

can

infer

structure, ratios

were

two

equivalents

titration

was

complete

titration

accounted

for

analytical

the data

for

N’-(S-amino

the

group. for

one

X4 shows

using

aliquots

I shows

the

results

That

the

analyses

X4’

of

5-carboxypentanyl)-lysine

shown

in the

of a group

table.

with

an apparent

performed

using

consumed

60 micro-equivalents

nitrogen

performed

hygroscopic

performed

Table

a structure

also

of X4 were

Because

mg of N in 25 ml of water.

The

of the

nitrogen

et -- al.

+carboxypentanyl)-lysine.

was

content

obtained

for

Accordingly,

X4

pK at pH

in an amount

providing of NaOH.

of X4. X4 were the

consistent

with

chemical

the

synthesis

undertaken.

of Nz(S-amino

5-carboxypentanyl)-lysine

was

accomplished

by the

scheme

1.

Comparison pounds

all

groups

supported

a peak

theoretical

10.2.

of NE-(5-amine

Synthesis shown

juncture,

revealed

the

nucleus

data

NMR

shows

showed

satisfactorily

of an aromatic

to an electron-withdrawing

and

postulated

and

showed

adjacent

of lysine

which

the

of N,

groups this

the

of X4

unequivocally

atoms

that

weighing

from

with

spectm

with

0.78

with

by comparison

Thus

of the

accurate

consistent

NMR

of both

(1929)

as ratios

absence

COMMUNICATIONS

.

containing

4

analyses

Slyke

the

acid

the

on carbon

lysine 7

RESEARCH

presence

determinations

by the

triplicate

compared

at 2.8

Manometric

way,

-N.

the

Where

peak

confirmed

of protons in CH2

The

of a solution

and

occur indicating

a corresponding

groupings

BIOPHYSICAL

in trifluoroacetic

In a positive

dinitrophenylation

significant

spectra

presence

as would

NMR

The

1965).

and

AND

to be identical

of X4 with with

N “-(5-amine

respect

5-carboxypentanyI)-Iysine

to chromatographic

577

behavior,

showed electrophoretic

the

two

mobility,

com-

Vol. 21, No. 6, 1965

BIOCHEMICAL

.TABLE I.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MANOMETRIC

Ratio

OF X . Theoretical for N ‘-(5-amine 5-carboxypentanyl)~lysine

Found

Total Nitrogen* Total Amino Nitrogen**

1.61

1.50

Total Amino Nitrogen** Total a-Amino Nitrogen**”

1.02

1.00

Total Carbon Total Nitrogen’

3.95

4.00

6.13

6.00

Total Carbon Total a-Ammo

*

dinitrophenylation, In contmst, (4amino

Nrtrogen**”

Kjeldahl method (pM/ml) Nitrous acid-N2 method (PM/ml) Ninhydrin-C02 method (PM/ml)

** ***

spectml

(lysinoalanine)

consistent

from N s-(4-amino

ammonia,

whereas,

et -- al. (1964) and Miller

vestigators,

for contents

or N6-

N”-(2-amine

as described

difference

between

P-carbo-

by Bohak (1964), X4 and lysino-

is that the former does not react

lysinoalanine

in experiments

et -- al. (1964) in which Table

by this treatment

prepared

yields

after those of Partridge

it was shown that the desmosines derive from

and, at various

by the method of Miller

of lysine, isodesmosine, an increase

modeled

II shows the results of an experiment

embryos were incubated

there occurred

A further

proposed structures,

of X4 was studied

Aortas were removed, elastin analyzed

from X4.

in the infmred.

and glyoxal.

lysine residues in a “pre-elastin”. Hope chick

behavior

4-carboxybutanyl)-lysine

from ribonuclease

as shown by Bohak,

formaldehyde

and spectml

were also synthesized.

was prepared

distinguished

The biosynthesis

old Mount

which

with their respective

sodium periodate

lysine,

of DNP derivatives

4-carboxybutanyI)-omithine

and it too was readily alanine

behavior

X4 could be distinguished

xyethyl)-lysine

with

ANALYSES

periods,

IO day

a group was sacrificed.

et -- al. (1964), and the protein

desmosine and X4.

As shown by the other in-

in desmosine and isodesmcsine

578

in which

contents with

increase

Vol.

21,

No.

BIOCHEMICAL

6, 1965

AND

BIOPHYSICAL

FIGURE

RESEARCH

COMMUNICATIONS

1

r Constant pH 3.0 hl-bromosucclnlmlde

fH2-(CH2)3TH-COOH NH .Ac

NH2 Na-acetyl

1 O=CH-(CH2)3CH
--

I NH.Ac

-i

lysine

N-acetyl-a-amino

1

NH.Ac

reduction

HOOC
OF CHICK

AGE

Lysine

(6N

Pd)

HCI

at 108O)

NH2

!%carboxypentanyl)-lysine

OF

H2 over

I

NH2

DETERMINATION

I

(2) hydrolysis

!

II.

semialdehyde

I

(1)

TABLE

adipic

/

HOOCCH-(CH2)3CH2-N=CH-(CH2)3CH-COOH I NH.Ac [ Schiff Base

NE-(5-amino

1

OF

(Lysinonorleucine)

LYSINE,

DESMOSINES

EMBRYOS

AND

EMBRYOS

OR

AND

OF OF

(x

X4

IN

)

ELASTIN

OF

AORTAS

CHICKS

CHICKS

IO Day

12 Day

I5 Day

I7 Day

6 MO.

10.3

8.2

6.8

4.6

2.4

lsodesmosine

2.1

(0.52)

2.5

(0.62)

3.0

(0.74)

3.4

(0.82)

4.8

(1.2)

Desmosine

2.2

(0.55)

1.9

(0.50)

2.4

(0.60)

2.9

(0.72)

4.8

(1.2)

x4

0.53

1.0

(0.50)

(0.27)

0.63

* The numbers in parentheses acid residues, of detmcsine, numbers indicate leucine

(0.32)

0.72

(0.36)

0.84

(0.42)

indicate residues Per 1000 total amino isociesmosine or X whereas the other A’ equivalents in the nlnhydrin determination,

579

Vol. 21, No. 6, 1965

in age of embryo,

BIOCHEMICAL

and concurrently

that the contents

of X4 in e&tin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a decrease

of lysine.

also increased

Significantly,

with age of embryo,

the data revealed at a rate quite similar

to that for the desmosines.

DISCUSSION The properties elastins

of a newly

have been studied

the formula,

detail

as the content ported

5-carboxypentanyl)-lysine that was synthesized.

pattern

the evidence

of the desmcsines,

of lysine residues decreases.

independent

studies with

radio-lysine

is cogent that NE-(5-amine

desmcrsines, is a crosslinking Chemically, carboxyethyl)-lysine

and compared

and indeed

Partridge,

a function

the al-

communication,

5-carboxypentanyl)-lysine,

of

with age of embryo

that show lysine to be a precursor

prepared

5-carboxypentanyl)-lysine

has re-

of X4.

Thus,

in the manner of the

is related

by Bohak (1964) by c h emical

This substance

of designation,

one could then refer to X4 as lysinonorleucine.

was given the trivial

of an s-amino

aldehyde

with the s-amino

reduction

of the Schiff base.

analagous

reactions

modification

Experiments

occur in formation

and Mrs. Sheryl Devine

are pkmned

580

and degradation

of

Chemically,

through

to determine

of lysinonorleucine

for her excellent

2-

Using this system

group of lysine to an aldehyde,

of Michigan

to N s-(2-amine

name, lysinoalanine.

group of a second lysine molecule

We wish to thank Dr. Karabatsos analyses,

from a

amino acid in elastin.

N e-(5-amine

by oxidation

in content

in a personal

ribonuclease.

is synthesized

with

is indistinguishable

of chick embryos, show that it follows it increases

pre-

features consistent

Studies on the appeorance,as

i.e.,

of purified

with those of synthetically

that X4 has analytical

time, of this amino acid in the aortic elastin ready established

SUMMARY

amino acid, X4, found in hydrolysates

The data indicate

Ne-(5-amine

sample of this compound

described

in analytical

Pared model compounds.

AND

coupling

of the

a Schiff base, and whether

biochemically

in elastin.

State University technical

this compound

assistance.

for aid in the NMR

Vol. 21, No. 6, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES Bohak, Z., J. Biol. Chem., 23-9, 2878 (1964). Fmnzblau, C., Sinex, F.M. and Faris, B., Nature, 205,802 (1965). Miller, E .J ., Martin, G.R. and Piez, K.A., Biochem. Biophys. Res. Commun., n, 248 (1964). Partridge, S.M., El&n, D.F. and Thomas, J., Nature, 197, 1297 (1963). Partridge, S.M., Elsden, D.F., Thomas, J., Dotfman, A., Tesler, A. and Ho, P.-L., Biochem. J., E, 30C (1964). Sanger, F., Biochem. J., z, 507 (1945). Spackman, D.H., Stein, W.H. and Moore, S., Anal. Chem., 30, II90 (1958). Thomas, J., Elsden, D.F. and Partridge, S.M., Nature, 200,6’;r(l963). Van Slyke, D.D., J. Biol. Chem., 2, 425 (1929). Van Slyke, D.D., Dillon, R.T., MacFadyen, D.A. and Hamilton, P., J. Biol. Chem., 12, 627 (1941).

581