A comparative study of recent assays for the determination of cystine aminopeptidase (oxytocinase)

263

Clinica Chimica Acta, 54 (1974) 263-268 0 Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CCA 6402

A COMPARATIVE DETERMINATION

STUDY OF RECENT ASSAYS FOR THE OF CYSTINE AMINOPEPTIDASE (OXYTOCINASE)

T. VAN BUUL and A.P.M, V’AN OUDHEUSDEN Department

(Received

of Clinical

January

Chemistry,

Sint Jozef

Ziekenhuis,

Doetinchem

(The

Netherlands)

30, 1974)

Summary A simple and rapid calorimetric determination for oxytocinase in pregnancy serum is described. The assay which uses S-benzyl-L-cysteine-p-nitroanilide as substrate is based on the formation of an azo-dye. The method requires 5 min incubation at 25”, followed by 6 min colour development. Optimum conditions, K, determination, the relationship between enzyme activity and enzyme concentration, and the effect of time on enzymatic activity are described. The coefficient of variation of the proposed calorimetric method is 1.3%. The assay is compared with two calorimetric methods using L-cystine-bis-pnitroanilide as substrate and with the kinetic method using S-benzyl-L-cysteine-p-nitroanilide as substrate.

Introduction Interest in the determination of oxytocinase in pregnancy serum is still increasing. For routine purposes we prefer the kinetic method. Nevertheless there are still recent publications on calorimetric methods using L-cystine-di-flnaphthylamide [ 11 and L-cystine-bis-p-nitroanilide [ 2,3] as substrates. Methods based on L-cystine-di-fl-naphthylamide have several disadvantages from theoretical and practical points of view [4]. Therefore the kinetic methods with the p-nitroanilides have been developed [ 4-61. However, if oxytocinase is assayed calorimetrically, the method using S-benzyl-L-cystine-p-nitroanilide in 2-methoxyethanol is preferred, because this substance is more rapidly split by oxytocinase than L-cystine-bis-p-nitroanilide. The proposed method is based on the diazotization of the p-nitroaniline, which is liberated by enzymatic hydrolysis, followed by conversion into an azo-dye through coupling with N-(1-naphthyl)-ethylenediamine.

Materials and Methods A comparison of the following four methods was carried out: (I) A calorimetric method using S-benzyl-L-cystine-p-nitroanilide in 2-methoxyethanol as substrate. (II) A calorimetric method using L-cystine-bis-p-nitroanilide in 2-methoxyethanol. (III) A calorimetric method using L-cystine-bis-p-nitroanilide in 0.01 moles/l HCl with 2% Triton X-100 [3]. (IV) A kinetic method with the same substrate as method I [5]. Reagents (1) Substrates (I) Dissolve 58 mg S-benzyl-L-cysteine-p-nitroanilide (obtained from Boehringer Mannheim or BDH) in 50 ml 2-methoxyethanol (3.5 mmoles/l). (II) Dissolve 33.5 mg L-cystine-bis-p-nitroanilide (obtained from KochLight Ltd) in 10 ml 2-methoxyethanol (7.0 mmoles/l). (III) Dissolve 161 mg L-cystine-bis-p-nitroanilide . 2 HBr (obtained from Nutritional Biochemicals Corp.) in 50 ml 0.01 moles/l HCl with 1 ml Triton X-100 (obtained from J.T. Baker Chemicals) (5.0 mmoles/l). (IV) As I. (2) Buffers. I, II and IV: phosphate buffer 0.1 mole/l, pH 7.5; III: tris(hydroxymethyl)aminomethane buffer 0.25 mole/l, pH 7.7. (3) Sodium nitrite. 1.0 g/l in water. (4) Ammonium sulphamate. 5.0 g/l in water. (5) N-( 1-Naph thyl)-e thylenediamine . 2 HCl (obtained from J.T. Baker Chemicals). 0.2 g/l in 0.01 mole/l HCl. (6) HCl. 1.0 mole/l. (7) HC1. 2.0 moles/l. (8) HCI. 2.2 moles/l. The reagents must be as far as possible of p.a. quality. Procedure (I) Add 0.05 ml serum to 0.9 ml phosphate buffer. Pre-incubate at 25” for a few minutes. Then add 0.15 ml pre-incubated substrate I. Incubate for exactly 5 min at 25”. Stop the reaction by adding 0.5 ml 2.0 moles/l HCI. Add 0.2 ml 1.0 g/l sodium nitrite and mix well. Allow the tubes to stand for 3 min. Then add 0.2 ml 5.0 g/l ammonium sulphamate and mix. Allow the tubes to stand for a further 3 min. Add 3 ml 0.2 g/l N-(l-naphthyl)-ethylenediamine and mix. Colour development takes place immediately. The colour is stable for at least 4 h. The absorbance is read at 540 nm against a blank. The blank is the same as the determination of serum, except that first HCl and after that the substrate is added. (II) Add 0.1 ml serum to 1.0 ml phosphate buffer. Pre-incubate at 37”. Then add 0.05 ml pre-incubated substrate II. Incubate for 10 min at 37”. Stop the reaction by adding 0.45 ml 2.2 moles/l HCl. The rest of the procedure is as in I. The colour is stable for at least 4 h.

265

(III) Add 0.2 ml serum to 0.2 ml Tris buffer. Pre-incubate at 37”. Then add 0.2 ml of pre-incubated substrate III. Incubate for 10 min and stop the reaction by adding 1 ml 1.0 mole/l HCl. The rest of the procedure is as in I. The blank is made by using buffer instead of serum. The colour is stable for at least 4 h. (IV) Add 0.1 ml serum to 1.8 ml phosphate buffer and then add 0.3 ml pre-incubated substrate I; mix well. In a recording photometer the absorbance increment per minute is determined at 405 or 411 nm at 25” for 5 min. Results A bsorp tion spectrum The absorption maximum of the pink coloured azo-dye is 540 nm. For the kinetic method the wavelengths 405 or 411 nm are chosen, because at these wavelengths there is no absorption of S-benzyl-L-cysteine-p-nitroanilide and the cleavage product S-benzyl-L-cysteine [ 61. Enzyme kinetics Two sera of pregnant women, one with low and one with high activity, were diluted with serum of non-pregnant women and with NaCl solution 9 g/l. There is a linear relationship between enzyme activity and enzyme concentration in mixtures from serum of pregnant and non-pregnant women for the three calorimetric methods (Fig. 1, I-III). In mixtures of serum of pregnant women and NaCl solution 9 g/l, there is a slight deviation of the linear relationship for the serum with high activity (Fig. 2, I-III). The effect of time on enzymatic activity for the three calorimetric methods is demonstrated with three different sera as shown in Fig. 3, I-III. Calibration curves Standard curves for the four methods were prepared usingp-nitroaniline. In a cuvet 10 mm, Shimadzu Double Beam spectrophotometer UV 200 we find for the calorimetric methods at 540 nm: (I) at 25” AA/5 min X 353 = U/l; (II) at 37” AA/10 min X 90.9 = U/l; (III) at 37” AA/10 min X 48.7 = U/l. In a cuvet 10 mm, recording photometer Vitatron MPS we find at 25” : (IV) 405 nm AA/min X 2110 = U/l; 411 nm AA/min X 2849 = U/l.

Fig. 1. I-III. Relation between enzyme activity and enzyme concentration methods. Dilutions of pregnancy serum are made with non-pregnancy serum.

for the three calorimetric

266

jx

ix

.

ix

eniyme

Fig. 2. I-III. Relation between methods. Dilutions of pregnancy TABLE

$A

*

;Y

enzyme

enzyme activity and enzyme concentration serum are made with NaCl solution 9 g/l.

$x

x

for the three

enrylnne

calorimetric

I

THE INFLUENCE

OF THE TIME MEASUREMENT Activitv

Serum

(U/l);

incubation

ON THE COLORIMETRIC

METHOD

1

time

5 min with use of a stop-watch (factor = 353)

5 min with use of a stop-watch for the first determination only (factor = 353)

10 min with use of a stop-watch (factor = 176.5)

66 60 66 73 78 94 102 148

65 60 IO 71 78 95 102 148

65 61 67 72 80 91 100 143

III-$,+;,;;;p:;; jlr&4:: 5

Fig. 3. I-III.

10

15

5

rni”

Enzyme-time

2

4

Fig. 4. Enzyme-time

6

relationship

8

15

mm,"

10min

relationship

Fig. 5. Comparison of two ordinate: method I.

In

method

for the determination

10

Y---iPmn

methods.

50

for the calorimetric

methods

5

for the three calorimetric

100

150

U/l

I in the lower time region.

of serum oxytocinase.

Abscissa:

method

IV;

267

Coefficients of variation The coefficients of variation were determined for the three calorimetric methods. The results are: (I) % = 61 U/l; S.D. = 0.8 U/l; C.V. = 1.3% (N = 20); (II) X = 14.6 U/l; S.D. = 0.4 U/l; C.V. = 2.7% (N = 20); (III) X= 17.7 U/l; SD. = 0.3 U/l; C.V. = 1.7% (N = 16). The coefficient of variation (C.V.) of the kinetic method is 1.1% [6 1. Twenty determinations of one serum were performed by a routine technician using method I. The results are: X = 68 U/l; S.D. = 0.8 U/l; C.V. = 1.2%. There is a good agreement between the results of the determination by method I with the use of a stop-watch for each time of pipetting and those with the use of d stop-watch for the first determination only and after that pipetting successively (Table I). A slight deviation of the incubation time of 5 min causes a greater error than the same deviation of the incubation time of 10 min. Moreover, the incubation time of 5 min of method I may be too short for routine purposes. Therefore the incubation time can be prolonged until 10 min, because the enzyme-time relationship is linear up until 10 min, also for high activities (Figs 3-I and 4). The results obtained with an incubation time of exactly 5 min and those with an incubation time of exactly 10 min agree very well (Table I). K,

determination The curves obtained by plotting initial reaction velocity versus substrate concentration is typical for enzyme reactions exhibiting Michaelis-Menten kinetics. A Lineweaver-Burk plot of these curves gives a straight line. The K, values are presented in Table II. Comparison of the methods A comparative study was carried out between the kinetic method (3~~) and the three calorimetric methods on sera selected at random. The regression equations and correlation coefficients are given below. y,, calorimetric method I (Fig. 5). y, = 2.29 + 0.93 x4; r1 = 0.98; nl = 68; t = 40.61;~ < 0.001. y2, calorimetric method II. yz = 1.63 + 0.16 x4 ; r2 = 0.96; n2 = 68; t = 28.27;~ < 0.001. y3, calorimetric method III. y3= 1.67 + 0.14 x4 ; r3 = 0.98; n3 = 67; t = 40.31;p < 0.001.

TABLE THE

K,

Method

II VALUES

OF

K,

I

0.22

II

0.03

III

0.21

IV

0.22

THE

FOUR

in &noles/l

METHODS

268

The regression equation and correlation coefficient for method method III (x, ) are: y5 = 6.53 + 7.46 x3 ;rs = 0.97; n5 = 67; t = 32.66;~ < 0.001.

I (ys ) and

Discussion We have tried to show that the calorimetric method with S-benzyl-Lcysteine-p-nitroanilide as substrate can replace the kinetic method (using the same substrate), if no recording photometer is present. The advantage of the kinetic method is that the progress of the reaction can be followed during the measuring time and that no blank is required. The proposed calorimetric method is more sensitive than that with L-cystine-bis-p-nitroanilide as substrate, because S-benzyl-L-cysteine-p-nitroanilide is split more rapidly by oxytocinase. Therefore the measuring time can be reduced to 5 min. Moreover the reaction can be performed at 25”) because the activity is high. If the reaction is performed at 37”, the activity is about 2.5 times as high as at 25”. For this reason smaller differences of activity can be determined with methods I and IV than with methods II and III. The most accurate results will be obtained by method I if the incubation time of 5 or 10 min is measured exactly with a stop-watch for each determination. 2-Methoxyethanol is a better solvent than 0.01 mole/l HCl with 2% Triton X-100 [6]. The substrate solution of method III is turbid, whereas those of methods I, II and IV are clear. The incubation mixture of method III is turbid too, and becomes clear only after HCl is added. Further the serum blank of method III is not clear, so a buffer blank has to be performed, which is not correct. The serum blank of methods I and II are clear. However, if method III is performed with 2-methoxyethanol as solvent for the substrate, a clear substrate solution, then a clear reaction mixture and a clear serum blank result (Shimadzu Double Beam Spectrophotometer UV 200, cuvet 10 mm, 540 nm, 37”: AA X 48.1 = U/l). The K, value, given by Usategui-Gomez et al. for their method (III) is too high: 2.5 mmoles/l. We obtained a value of 0.21 mmoles/l. Acknowledgement We thank Miss A.R. Verhoef

for her skillful technical

assistance.

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