Coordination ability of the thyrotropin releasing factor. I. Interactions with cupric ions

INORG. NUCL. CHEM. LETTERS Vol.15, pp. 387-392 Pergamon Press Ltd. 1979. Printed in Great Britain

COORDINATION

A B I L I T Y OF THE T H Y R O T R O P I N

R E L E A S I N G FACTOR.

I. I N T E R A C T I O N S W I T H C U P R I C

IONS

G.Formicka-Koz~owska, H.Kozlowski, B.Je~owska-Trzebiatowska XG.Kupryszewski,*J.Przybylski I n s t i t u t e of C h e m i s t r y , U n i v e r s i t y of W r o c l a w , J o l i o t - C u r i e 14, 50-383 Wroc~aw, Poland ~ I n s t i t u t e of C h e m i s t r y , U n i v e r s i t y of G d a 6 s k , S o b i e s k i e g o 18, 8 0 - 9 5 2 Gda~sk, P o l a n d

(Received 16 August)

The bits

synthetic

the

volvement known

of

up

to

this

usual some

hormone its

ability. acid

as

the

metal

in

communication

are and

still on

TRF a s

in

to

to

examine for

ions

about

the

its

in

is

vivo

metal

This

dipeptide

is

pyro-

of

analogue

im-

quite

systems.

in

rare

extremely

interactions natural

exhino

quite

the

systems.

to

Though

promise

histidine

natural

beneficial

are

metal

available

or

as

the

TRF,

factor.

seemed

hand

us well

ions

data

other site

inspired

for

with

pattern

no

the

essential

Cu(II)

releasing

interactions

coordination

has

with

thyrotropin

residue

are

results

tamyl-L-histidine

the

There ion

which

the

acid

sequence

ions

of

amino

interaction

peptide metal

L-pyroglutamyl-L-histidyl-L-prolinamide,

activity

date,

coordination glutamic portant

tripeptide

biological

un-

TRF w i t h In

this

L-pyroglu-

presented.

EXPERIMENTAL TRF a n d in

L-pyroglutamyl-L-histidine

ref.(i).

i:i

Cu(ClO4/2.6H20

Cu(II):

TRF a n d

experiments.

The

Cu(II):

pH w a s

(Pyr-His)

from

Fluka

Pyr-His

adjusted

was molar

with

were

used

as

ratio

NaOH a n d

synthesized

the

metal

solutions

as

ion were

measured

described

source. used

The

for

all

on a Mera-Elmat

N 5i22

pH-meter. Absorption Circular

spectra

&ichroism

ropolarimeter

-PS-iO0

taken

the

on a Beckman

were

recorded

All

CD r e s u l t s

are

o n a JEOL J E S - M E - 3 X

1H NMR e x p e r i m e n t s

were

UV 5 2 4 0

spectrophotometer.

on an

automatic

expressed

in

spectrometer carried

out

at

recording

terms

of

liquid

o n a JEOL

A6

spect(Q-~.).

nitrogen

tem

1 0 0 MHz JNM-

spectrometer.

The lyte,

were

and

measured

spectra

JASC0-J-20.

EPR s p e c t r a perature

were (CD)

EPR s p e c t r a so

the

best

were

measured

resolved

for

spectra

solutions could

be

containing

0.5

M NaCIO 4 electro

obtained.

RESULTS AND D I S C U S S I O N The d-d t r a n s i t i o n

energies

lutions

at s e v e r a l

pH v a l u e s

the d-d

transition

energy

sequential

coordination

as ~ e l l

are

as the EPR p a r a m e t e r s

listed

for c u p r i c

in T a b l e

i. The

complexes with

of n i t r o g e n donors, 387

for C u ( I I ) T R F

stepwise

increasing

increase

soof

pH s u g g e s t s

as in the case of the o t h e r CuIIl)

388

Coordination Ability of the Thyrotropin Releasing Factor-l.

polypeptide Cu(II) N3

systems

(2-4). The s e l e c t i v e

ions are a d d e d

suggests

(imidazole nitrogen)

IH NMR p e p t i d e

that the metal

S p e c t r a and EPR D a t a

pH

A,nm

Cu(II) : TRF

I:I

£

758sh 617

28.1

6.32

602

49.4

6.80

599

61.6

8.32

595

71.3

g,

11.30

571

77.6

2.2i2

197.6

78.I

2.209

20i.8

568 i:i 590

51.I 57.9

11.39

570

54.5

EPR s p e c t r a

of C u ( I I ) T R F

five-line

tion at 599 nm are

superhyperfine

above

suggest

Consideration

are

appears

: TRF s y s t e m

supports

mentioned the G u p r i c

a b o v e and

The s p e c t r a l

that the three This

line b r o a d e n i n g w h e n Cu(II)

These

results

the most p r o b a b l e

and that n o n e system which

results

Ii. donor

N- of the p e p t i d e

of two p r o l i n e

residue

is also

ions are a d d e d

is the close a n a l o g u e

for b o t h s y s t e m s

coordinated

is not d i r e c t l y

conclusion

If

in the h i g h pH region.

the Cu(II) P y r - H i s

that p r o l i n e

complexes.

acid,

linkage.

in the pH r e g i o n a b o v e that

is the N3 of i m i d a z o l e ,

in c o o r d i n a t i o n

system.

the view

suggests

to the

to 570 nm and a s e v e n -

(A N = 16 G ) ( F i g . l ) .

c o o r d i n a t i o n of a third n i t r o g e n

studied

resolved

from two c o o r d i n a t e d ni-

and m o s t p r o b a b l y N- of the p e p t i d e

of the m o l e c u l a r m o d e l

involved

of the Cu(II) TRF

deriving

the c o o r d i n a t i o n of two n i t r o g e n s

and N- of the p y r o g l u t a m i e

%re h a v e also this

structure

ii the d-d b a n d p o s i t i o n s h i f t s

EPR s t r u c t u r e

in the i:l Cu(II)

nitrogens

in the pH r e g i o n 6-10 are w e l l

the EPR s p e c t r a as well as the d-d band posi-

consistent with

superhyperfine

linkage,

Thus

: N3 of i m i d a z o l e

the pH i n c r e a s e s

set

solutions

(AN = i6 G ) ( F i g . l ) .

strongly

188.0

73.3 75.6

585

-line

2.222

592

6.98

ion

182.9 190.1

585

10.14

cupric

2.268 2.223

9.50

Pyr-His

i : I

All , G

10.68

Cu(II):

trogens

I

for Cu(II) : TRF and Cu(II) : P y r - H i s

5.72

11.82

and show

the c o o r d i n a t i o n at

at p H ~ 4 . TABLE

Absorption Systems.

line b r o a d e n i n g w h e n

ion b e g i n s

donors

are

involved

supported

to a TRF

are s i m i l a r and

the n i t r o g e n s

in the f o r m a t i o n of

by the s e l e c t i v e

solution

IH NMR

in the h i g h pH re-

giono The ratio

spectroscopic

and EPR s t u d i e s

solutions

have not

1:2 m o l a r ratio

species.

indicated

of 1:2 Cu(II)

: TRF

(or Pyr-His)

the f o r m a t i o n of s i g n i f i c a n t

amount

molar of any

Coordination Ability of the Thyrotropin Releasing Factor-l.

389

~g=2.0023

FIG.

i

EPR S p e c t r a o f 1 : 1 C u { I I ) : TRF Frozen Solutions a t pH = 9 . 4 0 ( a ) and ll.40(b) .

Thus

the

formation

o f C u ( I I ) TRF ( o r

H # - CHz 0

/

Pyr-tIis)

complex proceeds

/Pro-NHz 0 CO H~-(~l-lz tl,-, / 0 C~. /'CHC "= N -CH O_EHN u" {HzO)~C~'/CHz / ,,

Pro-NH2

OC', .~:H-~-~-I~H-CO NH FI CHz I-Iz0., _ N/'I'~ ..Cu~ L ~ H H20 "HzO ( CuL}~

NH (CuH.1L)+

o.-JI.* /CI'~.

,/.0

iPro-NHz

H~/C~ ¢0 ,Pro-NHz ~it-- I CH-C~ ,-, - / . ,-,-, ~ L'U

~.c: ~c.-c~,_, .co O/,'L'~.'~N\

/N-CH

,,c< CH,

OH-

~N/.~\

,~'~ M

~OH-

M --U-I

o

--NN

[CuH_~L{OH-)]z-

[Cu H.zLIOH')]"

as

follows

390

Coordination Ability of the Thyrotropin Releasing Factor-l.

CD s p e c t r a in

Table

peak

at

phore

2.

of

i:i

about

of

the

2 3 6 nm d e r i v i n g metal-free

le

2).

from

suggests

proline

cis-trans

mophore

contribution

the

Cu(II)TRF Two o t h e r

ly

sensitive

rized the

to

band

Cu(II)

isomer

the

to

at

297

by

Cu(II)TRF

TRF c o m p l e x

~ith

system is

most

terminal

the

Cu(II)Phe-Gly-Gly

N.

are

found at

to

(8).

at

is

(8).

transfer

in

ion of

the

bulky

supported

Cotton amino

transitions

3i5

positive are

TRF.

one

in-

T h e CD s p e c t r a are

signs

characte-

to

those

in

the

on

pattern

most to

of

Cu{III

mainly

CD s i g n

nitrogens

peak

slight-

of

residues,

nm CD b a n d s

in

chelates.

: the

effect

from peptide

chro-

observed metal

opposite

similar

(Tab-

features

to

acid

by the

2 8 3 nm a n d

the

i4%

on the

consists

signs

about

carbonyl

tripeptides

with

chromoat

ion

ion

CD p e a k s

region

studied

of

Cu(II)

in

positive

peak

extent metal

Both

this

but

of

its

The

of

presented

the

carbonyl

proline

coordination

the

3 0 0 nm, to

the

with

rotation 2).

are {a)

negative

the

on the

enhancement

:

C u ( I I ) TRF s o l u t i o n s

The reversing due

as

spectrum

about

to

system

3 1 5 nm ( T a b l e

majority

conclusion case

for

the

coordinated

restricted

copper(II)

the

the

the

well

The

of

(b)

present

in

of as

more

peak

probably

This

from charge

remain

to

and

isomer

CD s i g n .

peaks

n~Xtransition

influence

due

extrema

the

derive

the

is

nm d u e

t~o

proline

The absorption

complexes

also

the

systems

b y two

group)(5-6)

equilibrium

negative

pit.

from

Both peaks

UV f e a t u r e s

2 8 3 nm a n d

tense

eis

a minor

complex

at

of

the

: Pyr-His

characterized

prolinamide

TRF ( 7 ) .

This

: TRF a n d C u ( I I )

TRF i s

2 1 2 nm d e r i v i n g

(mainly

in

Cu(II)

A metal-free

in

probably

the

cupric

ion.

In the visible region a symmetric the Cu(II)

positive

peak at 588 am is observed

complexes with two coordinated nitrogens

from the Big--* B2g transition within 405 nm could originate

the Cu(II)

(see above).

effects

at 572 nm and 475 n m ; they arise

sitions. Cu(II)

two bands are observed,

The positive

complexes

sign of the former

with peptides

nal amino acid residue pyroglutamio (i.e.

three coor-

and negative

transition

is characteristic

the bulky side-chain

for the positive

rule. Thus

sign of the hand at 572 nm. from II to above 12

band at 368 nm) could be caused by the

of the imidazole Ni nitrogen,

N3 nitrogen of the histidine

only for

in the N-termi-

(9), and is in agreement w i t h the hexadecant

of the positive

Cotton

from Big---> B2g and Blg---~ Eg d-d tran-

of the CD spectrum as the pH is increased

the appearenoe

deprotonation

complex with

with positive

containing

acid is responsible

Small variations

band at

though its position seems

to be too far from that at 588 nm. For the Cu{II)TRF dinated nitrogens

This derives

ion. A small positive

from the Blg---~ Eg transition,

for

residue would

as the Cu(II) promote

coordination

such a d e p r o t o n a t i o n

to the at

high pH (I0,II) . The CD results

for Cu(II) Pyr-His

Cu(II) TRF and support

system are almost

the above mentioned

in the latter system has only a minor tion in the Cu(II) TRF complex.

conclusion

influence

identical

as those for

that the proline residue

on the symmetry and conforma-

Coordination Ability of the Thyrotropin Releasing Factor-l.

391

TABLE 2 Circular D i c h r o i s m of Cu(II) C u ( I I ) : TRF 1:1

pH 6 . 0 0

- 9.30

A,nm

a£

588

+0.93

405 328 289 230 208

+0.01 -1.52 +1.35 -4.17 +4.70 metal-free

TRF t

pH = 1 1 . 2 5

Cu(II)

+1.09 -0.11

318 283 234 210

-0.73 +0.97 -3.86 +4.62

A,nm

: Pyr-His

pH = 1 1 . 0 9

587 418 331 290 233 205

+0.95 -0.005 -1.13 +1.04 -2.26 +6.93

Pyr-His,

metal-free

~£

574 470 326 287 238 205

A,nm

+3.71

Ag

572 474 319 284 238 207

+I.19 -0.i0 -0.52 +1.14 -2.98 +5.80

12.00 4£

210

+5.38

supported

No 1 0 . 4 . 2 . 0 1 . 5 . 2

A,nm

+1.15 -0.06 -0.75 +1.20 -3.01 +6.18

pH 1 0 . 2 0 A£

This work was p a r t i a l l y

grants

pH = 1 1 . 9 4

A,nm

pH = 6 . 7 8

research

-0.11 +2.24

i:i

A£

210

+1.12 -0.09 +0.05 -0.61 +0.96 -3.86 +4.09

5L

236 212

pH 6.00 - 9.20

A,nm

6£

572 474 368 315 283 235 211

pH = 1 0 . 2 0 -0.13 +1.94

A,nnl

A,nm

&£

572 475

hE

236 212

pH = 1 2 . 0 2

A,nm

pH = 7 . 1 0 h,nm

Complexes w i t h TRF and Pyr-His.

by the Polish Academy

of Sctenoes

and MR-I-9.

REFERENCES 1.

E . M A L I ~ S K I , J.NAUMANj J.PRZYBYLSKI, M.RYZECKA, J.SZAFRANEK a n d

2.

G.FORMICKA-KOZLOWSKA, H.KOZLOWSKI a n d B.JEZOWSKA-TRZEBIATOWSKA,

G.KUPRYSZEWSKI, P o l . J . M e d . P h a r m . Inorg.Chim.Aetaj

25,

1 (1977);

(in press) H.KOZLOWSKI, I n o r g . C h i m . A o t a ,

3. M . L . B A I R a n d E.M.LARSEN, J . A m . C h e m . S o e . , 4.

T.F.DORIGATTI and E . J . B I L L O ,

93,

1140

J.Inorg.Nuel.Chem.,

31,

(1971) 37,

1515 ( 1 9 7 5 )

5 . W.VOELTER, K.ZECH, P.G~BEL, O.OSTER a n d A.ATTANASIO, Z . N a t u r f o r s e h , 6.

142 ( 1 9 7 5 ) P.PRADELLES, J . V I C A R ,

135(i97~

30h,

J.L.MORGAT, S°FERMANDJIAN t K.BLAHA a n d P.FROMAGEOT,

Collect.Czeeh.Chem.Commun.,

42,

79

(1977)

392

7.

Coordination Ability of the Thyrotropin Releasing Factor-l.

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-

LAGRANGE, A.M.BELLOCQ and I . C . P . S M I T H , Febs

8, J.M.TSANGARIS, J.W.CHANG and R.B.MARTIN, J.Am.Chem. S o o . , 9 1 , 9. J.M.TSANGARIS and R.BoMARTIN, J.Am.Chem. S o o . , 9 2 , 4255 (1970) 10. R.J.SUNDBERG and R.B.MARTIN, Chem. R e v . , 74, 471 (1974) 11. T.P.A.KRUCK and B.SARKAR, Can. J . C h e m . , 5 1 , 3563 (1973)

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