205

Clinica Chimica Acta, 0 Elsevier/North-Holland

CCA

72 (1976) 205-210 Biomedical Press,

Amsterdam

-

Printed

in The

Netherlands

8012

y-GLUTAMYL-3-CARBOXY-4-NITROANILIDE: CHOICE FOR ROUTINE DETERMINATIONS TRANSFERASE ACTIVITY IN SERUM?

LIV THEODORSEN

a and

a Clinical Laboratory, Chemistry, Institute (Received

April

12th,

JOHAN

H. STR@MME

THE SUBSTRATE OF y-GLUTAMYL-

OF

b

the Norwegian Radium Hospital, Oslo 3, and b Division of Clinical of Medical Biology, University of Tromsd, 9012 Tromsm (Norway) 1976)

Summary y-Glutamyl-3-carboxy-4-nitroanilide has been tested as donor substrate in the assay of y-glutamyltransferase activity in serum, glycylglycine being used as acceptor substrate. This donor substrate is highly soluble even in neutral solutions, in contrast to the commonly used y-glutamyl-4-nitroanilide. The enzyme which apparently acts according to a ping-pong bi bi kinetic mechanism, shows an absolute KIL? value for y-glutamyl-3-carboxy-4-nitroanilide of about 0.64 mmol/l, and for glycylglycine of about 13.4 mmol/l. The former K1, value is significantly lower than that previously found for y-glutamyl-4-nitroanilide. The carboxyl derivative exhibits a marked competitive inhibitory effect on the y-glutamyltransferase. This effect is more pronounced than that of y-glutamyl-4-nitroanilide. The carboxyl derivative has somewhat higher absorbance in the range of wave length (400-420 nm) used to monitor the formation of the product. It is concluded that as donor substrate in the assay of y-glutamyltransferase activity of serum, the new derivative is not substantially superior to the y-glutamyl-4-nitroanilide conventionally used.

Introduction The activity of y-glutamyltransferase (GT, EC 2.3.2.2) of serum is now commonly measured in clinical chemical laboratories. GT shows a low degree of substrate specficity. Various natural and synthetic substrates have been used for the determination of its activity. However, most of the present methods in daily use are based on the donor substrate y-glutamyl-4-nitroanilide (glu-4-NA) and the acceptor substrate glycylglycine (Gly-Gly) [1,2,3]. The choice of final reaction conditions with these substrates is limited by the low solubility of

206

glu-4-NA and by the fact that both substrates and the product 4nitroaniline may inhibit the enzyme [4]. The reaction, therefore, has to be carried out at “suboptimal” substrate concentrations. Several derivatives with greater solubility than glu-4-NA have been synthesized, some of which might conceivably be suitable substrates in the assay of GT activities [ 51. Recently a kit using y-glutamyl-3-carboxy-4nitroanilide (glu3-CA-4-NA) as donor substrate has become commercially available (Boehringer Mannheim GmbH, diagnostica). Since limited information on glu-3-CA-4-NA as substrate for GT has appeared in the literature, we here present kinetic data relevant to the use of this substrate in the routine determinations of GT activity in serum. Materials

and methods

LGlutamyl-3-carboxy-4-nitroanilide (glu-3-CA-4-NA) and 5-amino-2-nitrobenzoate Calibration Solution were gifts from Boehringer, Mannheim GmbH, Biochemica Werk Tutzing, D-8132 Tutzing/obb. L-Glutamyl-4-nitroanilide (glu4-NA) and glycylglycine (Gly-Gly) were purchased from Sigma Chemical Co., St. Louis, U.S.A. Pools of human serum were used as the source of the enzyme. The GT activities of these pools ranged from 30 to 480 U/l when assayed under conditions recently recommended by the Scandinavian Society for Clinical Chemistry and Clinical Physiology [3]. This method also served as reference method in the present study. The GT-catalyzed formation of 5-amino-2-nitrobenzoate at 37°C from glu3-CA-4-NA was monitored photometrically at 408 nm for the first l---2 min. The absorbance of the product at this wave length was found to be 4% less than that of 4nitroaniline. The final reaction mixture contained per liter: Tris/HCl 100 mmol (pH 7.6-7.7 at 37OC!), MgCI, 10 mmol, glu-3-CA-4-NA 4 mmol, and Gly-Gly 100 mmol, and a volume fraction of serum as in the reference method [3]. These conditions are essentially the same as those in the commercially available kit from Boehringer. Deviations from the standard conditions are stated in the figure legends. The assays were carried out either in an LKB 7600 Enzyme Analyzer using 100 ~1 glu-3-CA-4-NA in water as starting reagent, or in a GEMSAEC Fast Analyzer (initi.al reading 15 s, reading intervals 20 s, number of readings 4-8) using serum as starting reagent. Results

and discussion

The GT activity measured with glu-3-CA-4-NA and Gly-Gly in Tris buffer showed a broad pH curve with pH optimum in the range 7.6-7.9 (37”C), i.e. apparently identical to that found with glu-4-NA as substrate [ 31. The monitored product, 5-amino-2-nitrobenzoate, has an absorbance maximum at 380 nm. As is the case when measuring 4nitroaniline, it is however necessary to record at higher wave lengths (400-420 nm), where the blank absorbance due to the substrate is low enough to give favorable signal to noise ratios. A disadvantage of the carboxyl derivative as compared to glu-4-Na is its higher absorbance at 400-420 nm (Fig. 1). This difference is more marked at

207

440

420 Wave Fig.

1.

(37OC).

Absorption Curve

length

spectra

1, Glu-3-CA-4-NA

of

(nm) glu-3-CA-4-NA (4 mmol/l).

and Curve

glu-4-NA 2, Glu-4-NA

in

the

incubation

mixture

without

serum

(4 mmol/l).

37” C than at 25°C since the absorption curve is displaced slightly toward longer wave lengths by increasing temperature. Fig. 2A and B show that under highly suboptimal substrate concentrations double-reciprocal plots of the activities vs. the substrate concentrations at different fixed concentrations of the second substrate demonstrate families of apparently parallel straight lines. This pattern is consistent with a ping-pong kinetic mechanism of enzyme action [6]. Fig. 2 (insets) allows one to calculate the absolute Kn? values for the substrates. From 9 different experiments the average absolute Khq values for glu-3-CA-4-NA and Gly-Gly were found to be 0.64 * 0.15 (S.D.) mmol/l and 13.4 + 2.2 (S.D.) mmol/l, respectively. The corresponding absolute K, values with glu-4-NA and Gly-Gly as substrates have previously been found to be 1.09 mmol/l and 15.8 mmol/l [4]. The theoretical maximum velocity was found to be about the same with the two substrates. In a higher concentration range of the two substrates the double-reciprocal plots show non-parallel lines (Fig. 3A and B). The slope of the apparent linear parts of the curves increases with increasing concentrations of the non-variable substrate. Moreover at the higher concentrations of the variable substrate a marked inhibition is found as shown by the upward deviation of the curves (Fig. 3). The Lineweaver-Burk plots presented in Fig. 3 represent a characteristic pattern for competitive substrate inhibition by both substrates of an enzyme acting according to a ping-pong bi bi kinetic mechanism [6]. We have previously found a similar pattern when using glu-4-NA and Gly-Gly as substrates [4]. However, the competitive inhibitory effect of the carboxyl derivative is more pronounced than that of glu-4-NA. Thus, the inflection point (the highest activity) of the inverted plot of Fig. 3A at a fixed concentration of 50 mmol/l GlyGly occurred at about 3.5 mmol/l of glu-3-CA-4-NA. With glu-4-NA as the vari-

208

A

4

t

8

(mmol/l)

-1

0.15 0.20 0.30 0.60

,

1

0.1

L

0.3

1 ~ GLygly

(ml/L)-'

Fig. 2. Effect on GT activity of various substrate concentrations in the lower range. Pooled serum was used as source of enzyme. The activities were measured in triplets in a centrifugal analyzer. A. Effect of various glu-3-CA+NA concentrations (0.2-1.0 mmol/l) at four fixed concentrations (2, 3, 6 and 12 mmol/l) of Gly-Gly. B. Effect of various Gly-Gly concentrations (3.2-16.0 mmol/l) at four fixed concentrations (0.16, 0.20, 0.30 and 0.60 mmol/l) of glu-3-CA-4-NA. Insets show replots of the slopes (stippled curves) and the intercepts (solid curves) vs. the reciprocal concentrations of Glv-Gly (A) and giu-3-CA4-NA (B). A

0.03

B

300 0.02 ‘;

2 3

/

z 0.01

L

I

I

1.0

I

I

2.0

GLU_31CA-4-NA (mmol/L) -’

I

I

0.01 1 GlYglY

I

I

0.03 (mmol/l)

8

0.05 -1

Fig. 3. Effect of GT activity of various substrate concentrations in the higher range. Pooles of serum were used as source of enzyme. The reactions were carried out in an LKB 7600 Enzyme Analyzer using glu-3CA-4-NA dissolved in water as starting reagent. The final pH of the medium was 7.6-7.7 (37OC). Each result is the mean of 4 parallels. A. Effect of va;iw~s glu-3-CA-4-NA concentrations (0.5-15 mmol/l) at three fixed concentrations (50, 125, and 300 mmol/l) of Gly-Gly. B. Effect of various Gly-Gly concentrations (20-300 mmol/l) at three fixed concentrations (1, 4, and 10 mmol/l) of glu-3-CA-4-NA.

209

able substrate the same occurred at a concentration of about 7 mmol/l [4]. On the other hand, the inhibitory effect of Gly-Gly seems to be slightly less pronounced with the carboxyl derivative as donor substrate. Thus the inflection point at a fixed concentration of 4 mmol/l of glu-3-CA-4-NA and glu-4-NA occurred at about 150 (Fig. 3B) and 90 mmol/l Gly-Gly [4], respectively. Glu-3-CA-4-NA and Gly-Gly in concentrations of 6 mmol/l and ‘200 mmol/l were found to give maximum GT-activity of serum (Fig. 3). The substrate concentrations used by Boehringer (4 mmol/l glu-3-CA-4-NA and 100 mmol/l GlyGly) give only less than 2% lower activity than the maximal obtainable one. Human sera analyzed under the latter conditions showed on the average about 1% higher activities than those obtained with the Scandinavian recommended method [3]. In contrast, the quality control serum Seronorm (Nygaard & Co., Norway) which is fortified with GT from pig kidney shows on the average 36% lower activity with the carboxyl derivative. The day-to-day reproducibility of these two methods was found to be about the same, i.e. the relative standard deviation being 1.9% and 2.4%, respectively (3 sera (GT activity about 50, 90 and 170 17/l) analyzed in triplicate, 15 determinations). Conclusion The high solubility of glu-3-CA-4-NA in neutral solvents represents its main advantage over glu-4-NA as donor substrate in the routine measurements of GT activities in serum. The carboxyl derivative can be used as starting reagent even when dissolved in water. The acid solution necessary to be used when applying glu-4-NA, conditions which increase the spontaneous hydrolysis of the compound [3], can be avoided. Moreover, if serum is used as starting reagent, the danger of spontaneous precipitation of the supersaturated working solution of glu-4-NA will be avoided. However, the carboxyl derivative also possesses disadvantages. It shows stronger competitive inhibitory effect on GT. Therefore its higher solubility cannot be utilized to “optimize” the conditions of the assay. Another drawback is its higher absorbance in the range of wave length (400-420 nm) used to monitor the product. All facts considered we do not find the carboxyl derivative to be substantially superior to the conventional glu-4-NA as substrate for the assay of GT activity of serum. The latter substrate, the properties of which have lately been studied by many workers and which is commercially available from many different firms, was therefore preferred in the GT method recently recommended by the Scandinavian Society for Clinical Chemistry and Clinical Physiology [3]. Acknowledgement We gratefully acknowledge Sauren and Oddvar Gamst.

the

excellent

technical

References 1

Szasz,

2

Rosalki.

G. (1969) S.B.

and

Clin.

Chem.

Tarlow,

D.

15,

124-136

(1974)

Clin.

Chem.

20,

1121-1124

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of Anne

Marie

210 3 Recommended methods for the determination of y-glutamyltransfrrase in blood (79’76) Scand. J. Clin. Lab. Invest. 36. 119-125 4 Str@mme. J.H. and Theodorsen, L. (1976) Clin. Chem. 22, 417-421 5 Szasz, G., Weimann. G.. StLhler. F.. Wahlenfeld. A.-W. and Persijn, J.-P. (1974) Z. Klin. Chem. Klin. Biochem. 12, 228 6 Cleland, W.W. (1970) in The Enzymes, Vol. 2 (Boyer, P.D., ed.). pp. l-65, Academic Press. New York