Note: Descriptions are shown in the official language in which they were submitted.
~7075
~.
.
sackground
Enzyme substrates with naphthylamines as chromogenic
groups linked to other amino acids have been described in
-~ the literature for the aetermination of transferases and
proteases, such as y-glutamyl transpeptidase, lecuine
aminopeptidase, oxytocinase, and trypsin. Orlowski et al,
Clin. Chim, Acta, 7:755-760 ~1962), and references cited
therein. The determination of transferase and protease -
activity in human serum, urine, and tissues may have
diagnostic significance; for example, the assay of y-glutamyl
transpeptidase activity in human serum may be useful in the
diferential diagnosis of liver diseases, because the enzyme
activity is particularly high in obstructive jaundice and
liver cancer while lower activities are observed in viral
; hepatitis and liver cirrhosis. Orlowski et al, supra. See
also Rosalki et al, Ann. Clin. Biochem. 7:143 (1970). The
majority of studies with respect to y-glutamyl transpeptidase
determinations have been carried out using naphthylamines in
ormulating the substrates and, unfortunately, the products
~i.e., naphthylamines) are both toxic and carcinogenic,
presenting undesirable risks for general laboratory u~e.
Many of the enzyme assays commonly performed in
clinical laboratories are NADH linked; that is, they .involve
a series of reactions which ultimately result in the
reduction of nicotinamide adenine dinucleotide (NAD) to its
reduced form, NADH. The NADH is then detected spectrophoto-
metrically or fluorometrically. The more recent fluorometric
procedures have the characteristic advantages of simplicity,
; speed, and economy, and often have the further advantage oE
~0~37075
~greater sensitivity. Typically, a fluorometric NADH-linked
test involves the use of a filter fluorometer which directs
ultraviolet light at a wavelength of about 340 nm against
the surface of the sample and which measures the fluorescence,
or rate of change of 1uorescence at an emission wavelength
of about 465 nm.
Other references illustrating the state of the prior
art are patents 3,979,447, 3,862,011, 3,773,626, 3,5gl,458, ~;
3,878,048, 3,892,631, and Wildes et al, J. Am. Chem. Soc., ;~
95:8, 2610 (1973), and Bayley et al, Eur. J. Biochem. 56
(2), 455-65 (1975). ~'
Summary r
. '
This invention involves the discovery of certain
compositions of matter useful as enzyme substrates in the
fluorometric determination of transferase (or transpep~idase)
activity in homogenates and biological Eluids. Such substrates
are believed novel and are relat:Lve:Ly saEe. or labora~ory use. `~
An especLally important advantage is that such substrates
yield, upon cleavage by the en~ymes under investigation, '~
fluorogenic moieties which have peak fluorometric excitatlon
and emission values approximately those of NADH-linked ~ests
and, therefore, assays for fluorometrically detcrmlning trans-
' ferase or protease activ:Lty by the use o~ ch substrates may
be conducted with standard fluorometers usin~ the ~ame Ellters
lntend~d for convcntlonn'L N~DI;t-:l:lnked ns~ay9. ~.ehu~ 1
transferase such as '~-glutamyl transpeptidase may be measured
using the same fluorometric equipment and filters used for
conductLng as~ays oE other enzymes such as SGOT, SGPT, CPK,
LDH, and HBD.
In one particular aspect the present invention provides
a composition of matter for use as a substrate in a fluorometric -~
determination of transferase and protease activity selected
jl/ l' -3-
~A
~887~75
from the group consisting of
COR1
NHR3
~ `:
COR2
wherein each of R~ and R2 is -OH, -NHz, NHCH3, -NHC2Hg,
-N(CH3)2, -N(CzH3)2~ -N(CH3)(C2H5), -OCH3, or O(CH2~nCH3,
n i~ an lnteger of l through 4, and wherein R3 is an amino
acid moiety capable of being cleaved from the remainder of
said substrate in the presence of a transferase or protease :
having activity specific to that substrate.
In another particular aspect the present invention ;
provides a reagent suitable for use in a fluorometric
determination of transferase activity of y-glutamyl trans-
peptidase comprising a y-glutamyl derivative of 5-amino-
isophthalic acid, dimethyl ester, and salts thereof, an :
: acceptor of the glutamyl moiety when said derivative is cleaved
in the presence of y-glutamyl transpeptidase, and a buffer
for maintaining a pH within the range of 7.5 to 9Ø
In a further particular aspect the present invention .:
provides a fluorometric method for determining the activity
of transferases and proteases in samples of biological fluids,
comprising the steps of mixing and reacting a substrate and a
: sample of body fluid containing a transferase or protease
capable of cleaving said substrate, then exposing the mixture ~:.
to ultraviolet light having a wavelength within the range of
320 to 380 nm, and measuring the rate of change in fluorescence
at a wavelength within the range of 420 to 480 nm, said
substrate being selected from the group consisting of
COR1
~ NHR3
OR2
. I .
jl/ -3a-
, : . .
~087075
wherein each of R1 and R2 is -OH, -NH2, NHCH3, -N~IC2H5,
-N(CH3)2, -N(C2H5)2, -N(CH3)(C2H5), -OCH3, or O(CH2)nCH3,
n is an integer of l through 4, and wherein R3 is an amino
acid moiety capable of being cleaved from the remainder of
sald substrate ln the presence oE a transferase having
activity specific to that substrate. ..
In yet a further particular aspect the present invention
provides a fluorometric method for determining the activity
of y-glutamyl transpeptidase in a sample of biological fluid,
comprising the steps of mixing and reacting a y-glutamyl
derivative of 5-aminoi.sophthalic acid, dimethyl ester, or a
salt -thereof, with a y-glu~amyl acceptor, a buffer for
maintaining a pH within the range of 7.5 to 9.0, and a sample ::~
of biological fluid containing y-glutamyl transpeptidase, then
exposing the mixture to ultraviolet light having a wavelength
within the range of 320 to 380 nm, and measuring the rate oE
change in fluorescence at a wavelength with:Ln the range oE
420 to 480 nm.
. Description
The enzyme substrates involved in this invention
....
.
~ - .
~; jl/;`- -3b-
': I
~ ~87~'7S
are 5-aminoisophthalic acid derivatives of the general formula
CORl
~ NHR3
COR2
and wherein each of R1 and R2 is -OH, -NH2, -NHCH3, -NHC2H5,
I~(CH3)2~ -N(C2H5)2~ -N(CH3)(c2H5)~ -OCH3, or -O(CH2)nCH3, n is
an integer from 1 through 4, and wherein R3 is an arnino acid
moiety capable of being cleaved from the remainder of the su~-
strate when exposed to a transferase or protease having activity
specific to that substrate, in some cases in the presence of
glycylglycine or some other appropriate acceptor such as
glutamate, glycine, or glycylglycylglycine. Such substrates
which have amino acid moieties ~that may comprise s~veral amino
acid groups) and which are specific to various transferases and
proteases are as follows [in each instance the fluorogenic
moiety as identified above is represented by the designation (A)]:
Substrate Enzyme
5 ~ ~ ~ (A)-lys-ala DAP-II
(A)-Z-ala-arg-arg Cathepsin B 1
(A)-BZ-val-lys-lys-arg Cathepsin B la
(A 2-HCl)CBZ-arg-arg Cathepsin B 1
(A-diacetate)-N-CBZ-arg-arg-arg Trypsin
(A 3-HCl)-L-arg-arg DAP III
A)-Z-gly-gly-arg Anionic Trypsin, Plasminogen Activator,
Pro;nsul;n Converting Enzyme
(A)-pro-arg DAP-I or Cathepsin C
(A)-a-B'~-phe-val-arg Thrombin
~dl-A)-L-cystine Oxytocinase
(A)-y-g'lutamyl y-Glutamyl Transpeptidase
(A formate)-L-leu-gly-gly
(A)-leu Aminopeptidase
(A)-BZ-arg-pr'o-gly-phe-phe-leu Cathepsin D
(A)-phe-pro-ala-met Cathepsin B lb
(A)-glutaryl-gly-L-phe
(A)-gly-pro DAP-IV
(A)-CBZ-pro-ala-gly-pro Collagenase
(A)-his-ser DAP I or Cathepsin C
(A)-N-CBZ-L-pro-L-phe-L-his-L-
leu-L-leu-L-val-L-tyr-L-ser
(A)-N-CBZ-gly-L-met Renin
(A)-glutaryl-ala-ala Elastase
(A)-BZ-arg-pro-gly-phe-phe-pro Cathepsin D
(A)~ala Aminopeptidase B
(A)-BZ-arg Trypsin/Cathepsin B 1
(A)-BZ-arg-gly-leu
(A)-met
(A)-BZ-arg-gly-tyr DAP-I
(A)-ser-tyr Catheps;n C
-- 4 --
1~137~5
In the above, the designations constitute established
abbreviations as follows: ala (alanine), arg (arginine), BZ
(benzoyl), CBZ and Z ~carbobenzoxy), gly (glycine), his
(histidine), leu (leucine), lys (lysine), met (methionine),
phen (phenylalanine), pro (proline), ser (serine), tyr
(tyrosine), val (valine). To increase solubility rates, all
of the substrates may, if desired, be converted to salts
such as, for example, the hydrochloride, hydrobromide, acetate,
or formate salts of the amino acids.
Each of the substrates, when exposed to its
corresponding enzyme, is cleaved, the amino acid moiety being
released or coupliny with a suitable acceptor such as
glycylglycine, to leave the fluorogenic primary amine (i.e.,
substrate (~), as identified above, in which the substituent
for R3 is ~ hydrogen atom). All of such fluorogenic aromatic
amines have peak excitation and emission characteristics,
when exposed to ultraviolet light, which are sufficiently
close to those of an NADH-linked test (~ex=340 nm; ~em=465 nm)
to permit fluorometric activiky measurements using the same
equipment and filters employed for such standard NADH tests.
Specifically, such chromophores have peak excitation
charac~er~t.~cs at a wavelength within th~ rancJe o~ 320 to
380 nm and peak emission characteristics at a wavelength
within the range of 420 to 4B0 nm. For example, if substrate
(~) has methoxy groups as Rl and R2, then the resulting
chromophore will have a peak excitation wavelength of about
335 nm and a peak emission wavelength of about 445 nm.
In practicing the method of this invention, substrate
is first dissolved in a sterile aqueous solution which pre-
ferably contains a suitable buffer to insure that the pH will
be maintained at or near the optimum pH of the enzyme of
7075
interest. For example, where the enzyme to be measured is
~-glutamyl transpeptidase, the reaction may be measured over
a broad range of pH values from about 7.5 to 9.O, a pH of
8.2 ylelding maximum activity in the Eluorometric assay system
The substrate solution is mixed with the sample (suspension
or solution) and transferred to a suitable cuvet with any
suitable fluorometer being used to measure front-surface
~luorescence. The rate of production of the fluorogenic
compound is directly proportional to the amount of transferase
present in the sample.
The following examples are further illustrative of the
invention:
Example 1
Serum ~-glutamyl transpeptidase may be measured
fluorometrically by utilizlng r- tL-glutamyl) -S-aminolgophthalic
acid, dimethyl ester, hydrochloride salt, as the substrate
t Such substrate has the structural formula:
':
CH3O~C~O
~C~O
OC}I~
; The reagent solution contained 5 mM substrate, 55
mM glycylglycine, and 100 m~l Tris buffer (pH 8.2 at 25 C.),
thc solut:Lon volurlle belng 1.5 ml, The reagent solution was
warmed to 37 C., sample was added (volume at 0.05 ml), the
reactants were mixed and pumped lnto a flow-through cuvet.
T`he rate of increase in fluorescence was then measured for a
minimum of 4 minutes using a front-surface instrument
(~ex=365 nm; ~em=465 nm). By such a procedure, the rate of
change in fluorescence of the end product (5-aminoisophthalic
--6--
bm:~
, I
~0~7075
., .
acid, dimethyl e.ster) resulting fr~m the hydrolysis of the
substrate was measured, and the slope was calculated as the
change in fluorescence per minute of reaction.
Example 2 ;
The results of serum samples tested in accordance with
Example 1 were compared with the results of chlorimetric
~,, .
assays run on the same patient samples, using GGTP reagent
as marketed by Dade Division of American Hospital Supply
Corporation and following the method set forth in the package
instructions. To facilitate interpretation of data, the a
F/min. ~as changed to International Units per liter (IU/L? by ;~
to~aling IU/L and ~/F min. and deriving a factor IU/~ F.
The sera was tested in two groups o~ lbi, one group represent-
`:
ing undiagnosed conditions and the other diagnoscd conditions,
and the following results were obtalned:
~-Glutamyl Transpeptldase
Acti~ity (IU/L)
Sample _luorometrLc olorimetric
1 47 45
2 52 51
3 12.5 17
~ 85.4 79
113.4 110
6 196.9 195
7 14,5 17
8 l~i.5 18
9 3~5 3~
43.4 ~i8
11 40,8 72
3~ 12 236,~ 225
13 212.3 198
14 259.6 260
80.9 87
16 ~letastatic Cancer 87.5 119
17 Gastritis 89.5 94
18 Dehydratlon166.4 172 ;`
19 Obst. Jauntllce23 26
20 Colostomy 181 184
21 Hepatomegaly250.4 264
22 Cancer of Bladder 146.7 148
23 Jaundiced 164.0 167
24 Hip Proble~215.4 200
25 Hodgkins 14.7 14
26 Chest Pain, Hypertension 62.8 47
27 Pul~. Embolus174.2 150
28 Sarcodosis 193.5 178
- 7 -
- bm: -
- ':
.. , .. ; . . ,~ , , . ;,. ,; ~
37~
The data demonstrate excellent correlation between the
fluorometric method and the conventional colorimetric method
for the determlnation of serum levels of ~-glutamyl
transpept~dase. -
Example 3
The ~-(L-glutamyl)-5-aminoisophthalic acid, dimethyl
ester, hydrochloride salt, used as the substrate in Example 1
may be prepared by mixing phthalogy glutamic anhydride
(13.2 g, 0.051 mole) and S-aminoisophthalic acid, dimethyl
ester (10.4 g, O.OSO mole) in 60 ml of dioxane, and stirring
same at 55-60 C. (bath temperature) for 1.5 hours. After
evaporation of the solvent, the residue is then dissolved
in 200 ml of methanol and hydrazine hydrate (7.5 g, 0.15 mole).
The solution should then be flltered and allowed to stand at
room temperature (2 days). A resultlng whlte precipitate is
then collected, washed with 100 ml of water and 25 ml oE
! ethanol, a8itated in 100 ml of 0.5 N hydrochlorlc acid, and
filtered. The filtrate is treated with sodium bicarbonate
to give a pH of 6.5 to 7.0, and the precipitate (8 g) is
collected and dried. The hydrochloride salt may then be
prepared by dissolving 1 gram of the glutamyl derivative in
a fiolution of 0.3 ml of concentrated hydrochloric acLd and
6 ml of methanol. AEter evaporation oE the methanol, the
sol;Ld iB then dLrcd under reduced pressure.
Example 4
The following process may be used to prepare other 5-
amLnolsophthalLc acLd derivatives whLch may then be coupled
to appropriate amino acid constituents as indicated.
COOEI COOR C
(1) SOC12 ~ Hl ~ ~
~ N02 ~ ~/ \) ~ N02 ~--~3 ( \~ NEl2
/ (2) ROH ~ J ~ _/
COOH COOR COOR
bm~
.
... .... .... . .. .
~87075
.,
Where an amide is to be formed, RNH2 is substituted
for ROH in this equation. In either event, the end product
is then reacted with the particular amino acid desired
in the appropriate for~ (as illustrated in Example 3 in
connection with phthaloyl glutamic anhydride) to produce :
the final amlno acid derlva~ive of aminophthalic acid to
be used as a substrate for determining transpeptidase -
and/or protease activity. ~-~
While in the foregoing we have disclosed the
lQ invention in considerable detail for purposes of : -
illustration, it will be understood by those skilled in `~
the art that many of these details may be varied without
departing from the spirit and scope of th- invention.
'
".
~ .
.
'~
.1 ~
~ bm:~;J .
'
