Neuropeptides 3: 223-232, 1983

ENZYMATIC CLEAVAGE OF PYRDGLUTAMYL-HISTIDYL-DOPAkfNE , A TRH-

RELATED PSEUDO-PEPTIDE BY PORCINE SERUM AND BRAIN HOMOGENATE.

BY

Claude RICHARD,Arnold BOERSMA and Kia-Ki HAN ("I

Unite N" 16 de l'INSERM.Place de Verdun.59.045-LILLE cedex FRANCE.

and by

Elisabeth IMORIER and Richards RIPS.

Unite N" 98 de l'INSERM.17 Rue du Fer 5 Moulin.75.005-PARIS.FRANCE.

("):To whom all correspondences should be addressed.

ABSTRACT.

The Pseudo-peptide ( pGlu-His-Dopamine) was subjected

enzymatic degradation by porcine serum and brain homogenate.The

digests were quantitatively analyzed by HPLC to provide evidence

for lioeration of pyroglutamic acid and dopamine in serum and of

to

pyroglutamic acid in brain.The yield of liberation of pGlu is about

3 % in serum digests and about 0.70 % in brain digests after 3 minute

-s incubation.The time course of the yield of liberation of both

pGlu and dopamine in serum digests is determined.The "stability

in vitro" toward enzymes of serum and brain homogenate of a new

type of drug based on the combination of peatidic fragment of

TRH-(Thyrotropin-Releasing Hormone:pGlu-His-Pro-NH21 with a non

peptide moiety (dopamine) is considered and discussed.

223

INTRODUCTIM.

The significance of neuro-peptides such as TRH

(Thyrotropin-Releasing-Hormone) as chemical compounds that

provide an endocrine function in important neuro-physiological

processes is now well established.TRH was the first of the

releasing hormones to be isolated from porcine and bovine

hypothalamus tissue (l)(Z).TRH is a weakly basic tripeptide

( Pyroglutamyl-Histidyl-Prolinamide) and there are

three ring structures in the molecule, the pyroglutamic acid, the

imidazole ring of histidine and the pyrrolidine ring of proline. These

rings reduce the access to amide bonds and therefore the possibility

of enzymatic attack.

Rips and Morier (3) recently synthesized a pseudo-peptide consis-

ting of pGlu-His-Dopamine. These authors proposed a new type a drug

based on the combination of a peptide moiety (pGlu-His . ..> with a non

peptide moiety (Dopamine : 3,4-Dihydroxyphenyl-ethylamine). Morier (4)

suggested that a "carrier" might modify the pharmacological function

particularly the transport of a drug such as dopamine. This is one of

the reasons why these authors (3, 4) used a fragment of the TRH mole-

cule (pGlu-His . ..) as a "carrier" for the precursor of norepinephrine

and epinephrine, dopamine. However, it was not shown whether this

pseudo-peptide biological effects were due to the entire molecule or

to enzymatically products. This paper deals with the "in vitro"

enzymatic digestion by porcine serum and brain homogenate of this

pseudo-peptide.

MATERIALS AND METHODS

The TRH (pGlu-His-Pro-NH21 and the pseudo-peptide, pGlu-His-

Dopamine were synthesized and purified as reported previously by Rips

and Morier (3) and Morier (4).

Preparation of porcine serum : blood was collected and the serum was

separated and frozen for 20 min. The serum (10 ml) was diluted 6 fold

with 0.01 M phosphate buffer (pH 7.6) and dialyzed at 4°C for 24 h

against 1 liter of 0.1 M sodium acetate buffer (pH 5.5) ; the dialysis

224

tubing was spectropor-2 (5000-7000 MW). Following dialysis, the 10 ml

of serum was diluted to about 20-25 ml with water and lyophilized.

Preparation of brain homogenate : the brain was quickly removed, immer-

sed in 0.01 M phosphate buffer, NaCl 0.14 M (I:10 W/vol) and homogeni-

zed using a potter apparatus. The homogenate was centrifuged at 900 x g

for 30 min, the pellet discarded and the protein concentration of the

supernatant determined according to Lowry et al. (5). The protein

concentration in the 900 x g supernatant was adjusted to 10 mg/mL. The

supernatant was dialyzed against 0.01 M phosphate buffer (pH 7.6)

containing 0.14 M NaCL for 24 h at 4"C, 0.01 M phosphate buffer (pH

7.6), and 1 liter of 0.1 M of sodium acetate buffer (pH 5.51, before

dilution into 60 ml 0.01 M phosphate buffer (pH 7.0) and lyophili-

zation.

Degradation reaction : 4 mg of pseudo-peptide (IO JJM) was incubated

at 38°C with IO ml of serum (lyophilized powder redissolved in 5 ml of

0.01 phosphate buffer pH 7.35). The time course was established after

3, 30, 60, 120 min, 4 h and 8 h incubations. The reaction was stopped

by addition on an equal volume of 2 N HCl. After centrifugation at

3000 x g for 30 min, the supernatant was filtered again and 20 n1 'of

each sample were injected into the HPLC column. Enzymatic digests were

analyzed by the following methods. Pyroglutamic acid, dopamine, pGlu-

His-Dopamine, standard buffer, serum blank and brain homogenate were

detected by UV absorption at 206 nm of the eluent from HPLC (High

Pressure Liquid Chromatography). The HPLC was performed under our

original conditions : the column used was Microbondapack C-18 (Waters).

Elution was performed by a gradient from 0.1 per 100 trifluoro acetic

acid (TFA) to 0.1 per 100 TFA-acetonitrile (8:2 = V/V). The flow rate

was 1 ml per min and pressure was 1000 p.s.i. The total elution time

was 14 min. Pyroglutamic acid was from Sigma and Dopamine from Aldrich,

0.2 mg/ml of pyroglutamic acid, 0.2 mg/ml of dopamine and 0.25 mg/ml

of pGlu-His-Dopamine were dissolved in 0.1 per 100 TFA. 1 or 2 ~1 of

ach sample were injected in the HPLC column as standards.

225

RESULTS AND DISCUSSION

Our degradation reaction of synthetic pseudo-peptide is derived

from the conditions reported by Oliver et al. (6). We did set up an

original method of preparation of both serum and brain homogenate by

using exhaustive dialysis in order to eliminate free amino acids and

short peptides, which would prohibit further analysis of enzymatic

digests. The enzymatic activity of both serum and brain homogenate

before and after dialysis was controlled by the digestion of standard

TRH (pGlu-His-Pro-NH2). No significant difference of activity was

found in serum and only the diminution of 5 % of activity was found

in the brain homogenate.

Identification and quantification of enzymatic breakdown products

pyroglutamic acid, His-Dopamine and Dopamine were carried out by using

HPLC in a simple run.

Retention times of breakdown products and other components are

shown in Table 1.

TABLE 1 - RETENTION TIMES OF VARIOUS COMPONENTS ON THE HPLC Cl8

COLUMN EXPRESSED IN MINUTES AND SECONDS

pGlu 5 min

His 3 min 12 set

Dopamine 6 min 6 set

His-Dopamine 9 min 12 set

pGlu-His-Dopamine 12 min 24 set to 13 min

Serum "blank" 3 min 12 set to 3 min 48 set

Serum blank after 4 h autodigestion 2 min 48 set to 3 min 54 set (major peaks)

Brain "blank"

Brain "blank" after 4 h autodigestion

7 min 24 set 9 ; min and 10 min 24 set (minor peaks)

3 min 18 set and 3 min 48 set (major peaks)

5 min 18 set and 7 min 30 set (minor peaks)

3 min 18 see 3 ; min 48 set (major peaks)

4 min 30 set 5 ; min 18 set ; 7 min 18 set 8 ; min 6 set ; 9 min 6 set and 10 min 30 set (minor peaks)

226

Griffiths et al. (7) recently reported the inactivation of

Thyrotropin-Releasing-Hormone (TRH) by brain peptidases. These authors

did use HPLC for the identification of enzymatic degradation product

of TRH (7). The HPLC technique proved to be a valuable tool for

separation and quantitation of the yield of the pyroglutamic acid

and dopamine from the serum digested pseudo-peptide. This technique

not only provided evidence for the liberation of pyroglutamic acid

and dopamine but also allowed calculation of the yield of serum

breakdown products as a function of time (see Table 2).

TABLE 2 - THE YIELD OF LIBERATION OF PYROGLUTAMIC ACID AND DOPAMINE

FROM pGlu-His-Dopamine HYDROLYZED BY SERUM AS A FUNCTION

OF TIME

3 min 30 min 1 h 2h 4h 8h

pGlu 3.44 % 3.20 % 3.56 % 3.66 % 9.88 % 13.48 %

Dopamine 0.52 % 0.50 % 0.54 % 0.68 % 0.96 % 1.93 %

The half-time of TRH is relatively short in serum (8) and in brain

(9) (7). Interestingly the pGlu-His-Dopamine was cleaved and libera-

ted free pyroglutamic acidcabout 3 % in serum and 0.70 % in brain

after 3 minutes incubation (Table 2 and Table 3).

TABLE 3 - THE YIELD OF LIBERATION OF PYROGLUTAMIC ACID FROM pGlu-His-

Dopamine HYDROLYZED BY BRAIN HOMOGENATE AS A FUNCTION OF

TIME

pGlu

3 min 30 min 60 min

0.71 % 2.98 % 3.00 %

Taylor and Dixon (10) reported the existence of pyroglutamate

amino peptidase in rat serum. Matsui et al. (11) also reported the

presence of this enzyme in porcine brain extracts. In both cases, the

pyroglutamyl residue of TRH (pGlu-His-Pro-NH21 was removed yielding

His-Pro-diketopiperazine. For pGlu-His-Dopamine, in the serum enzyme

digestion, about 3 % in 1 hour, 4 % in 2 hours and about 10 % in

4 hours liberation of pyroglutamic acid and respectively about 1 %

227

liberation of dopamine in 4 hours digestion (see Table 2). However,

only about 3 % of pyroglutamic acid were liberated by brain extracts

without liberation of dopamine. The presence of a pseudo-peptide

metabolite, His-Dopamine was detected qualitatively in HPLC digestions

of pGlu-His-Dopamine (see figures 1 and 2).

The stability of peptide hormones and especially pseudo-peptides

in body fluids and tissues is an important consideration in terms of

duration of pharmacological action and biological half-life. Our

results provide evidence that pseudo-peptide undergoes biodegradation

"in vitro" yielding a small quantity of free dopamine (1 % after

4 hours incubation in serum). The mechanism of breakdown may be

explained as follows : both serum and brain extracts contain pyroglu-

tamate amino peptidase. Once the pyroglutamyl residue is removed by

pyroglutamate amino-epptidase, the remaining His-Dopamine is degraded

partially by aminopeptidase to yield free histidine and free dopamine

in serum digestion. The absence of amono-peptidase or low activity of

this enzyme in brain extracts is demonstrated by the absence of free

dopamine in brain extracts digestion of the pseudo-peptide (see

figure 4) (after 1 hour incubation). The autolysis of brain extracts

after 4 hours incubation makes further calculation of the digestion

of pseudo-peptide by brain extracts almost impossible, because the

autolyzed products of brain extracts did impair and possess the same

retention time of free dopamine (see figure 3).

In this paper, we have provided evidence of the relative

"stability" and rates of breakdown "in vitro" utilizing porcine serum

and brain extracts of pseudo-peptide containing Aopamine. Therefore

we can suggest that the pseudo-peptide possessing possible pharmacolo-

gical effects first reacts by their own action and then with extended

period of time might react as breakdown products His-Dopamine and

dopamine "in viva".

Such findings suggest that a better understanding of the mecha-

nism of degradation can provide a basis for the use of pGlu-His as

carrier with greater clinical potential ; our Paris group (Rips and

Morier) intends to more fully explor this aspect.

228

NEW- c

I

serum b

4 serum and

Stan

: 3min

0.D: 206 nm

Dopamine /

, Dopamine

Figure 1

lGlu.Hir. R Dopamine

0.D : 2ot

4

z 30 min

Figure-Z

229

u ZOOmin

:s2h

0.D 1206 nm

pGlu.His. I Dowmine

t -_4h

Figure-Z Bis

Figure-Z and Figure-2 Bis: Time course and eluti'on pattern of serur

digests of pGlu-His-Dopamine after 3 min.30 min.,60 min.2h and 4 h

incubation.

Figure 1: Elution pattern of serum blank and standard pGlu and

dopamine (Left).

Elution pattern of serum blank after 4 h autodigestion (Right).

230

0.D : 202 nm

Brain

Bl8Dk

Figure-3 Elution pattern of brain blank (left)

Elution pattern of brain blank after 4 h autodigestion.(Right).

Figure-4

D.D :2oenm

I I romin -

Elution pattern of brain digests of pGlu-His-Dopamine

after 3 min.,30 min., and 60 min. incubation.

231

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232