ANALYTICAL BIOCHEMISTRY 70, 181-186 (1976)

Automated Determination of Proteolytic Enzymes and of Amino Nitrogen by Use of Trinitrobenzene-

sulfonic Acid

C. A. ADAMS, T. C. ROBBERTS, AND K. C. BUTLER

National Food Research Institute, Council fi~r Scientific and Industrial Research, P.O. Box 395, Pretoria, 0001, South Africa

Received February 3, 1975; accepted July 16. 1975

Proteolytic enzymes are a s sayed using N ,N-d i me thy l casein as a subst ra te . The enzymes are determined in a homogeneous sys t em by measur ing free amino groups released. Proteolytic activity is expressed in gylcine equivalents . Amino nitrogen is assayed using an accelerated reaction with 2,4,6-trinitro- benzenesulfonic acid under alkaline conditions. The final reaction product is a sulfite adduct of the tr ini trophenylated amino group and its absorbance at 420 n m is measured . The procedure can also be used for the direct determinat ion of soluble amino nitrogen in the range of 25-250 nmoles/ml.

Since the early work of Okuyama and Satake (I), TNBS 1 has been used for the determination of amino groups (2) and for the assay of peptidase (3) and protease (4) activities. The rather lengthy incubation periods involved in these procedures precludes the use of automated systems. However, under alkaline conditions, the reaction time is con- siderably shortened (5). We have adapted this accelerated reaction for use in an automated system to assay proteolytic enzymes, and it may also be used as a direct assay for amino nitrogen.

MATERIALS Reagents

2,4,6-Trinitrobenzenesulfonic acid was obtained from B.D.H. Chemi- cals Ltd., Poole, England. N,N-dimethyl casein was prepared accord- ing to Lin et al. (4) but for best results we found it necessary to carry out the methylation twice on each batch of protein solution. N~N-dimethyl casein is also available from B.D.H. Chemicals Ltd., Poole, England, and has also been used by us but is often difficult to dissolve. Proteolytic enzymes tested were obtained from the following sources: Trypsin, Miles-Serevac Ltd., Cape Town, South Africa, and Pronase from Calbiochem, San Diego, Calif.

1 T N B S , 2,4,6-trinitrobenzenesulfonic acid.

181

Copyright © 1976 by Academic Press, Inc All rights of reproduction m any form reserved

182 ADAMS, ROBBERTS AND BUTLER

Equipment

The analytical system was constructed from standard Technicon Auto- Analyzer equipment. Glass transmission tubing was used wherever possible.

METHODS

Determination of Proteolytic Activity

Reagents. Borate buffer 0.05 M, pH 9.5. TNBS solution, 1.0% (w/v) in distilled water. The flask containing the TNBS solution is wrapped in aluminum foil. A volume of 50 ml is sufficient for several hours running time. Phosphate/sulfite solution containing 0.1 M NaH=PO4 and 0.002 M Na=SO3. The TNBS and phosphate/sulfite solutions are made fresh daily. N,N-dimethyl casein at a concentration of 5 mg/ml was dissolved in buffers appropriate for the enzyme being assayed. These were: trypsin, 0.1 M borate, pH 8.0; and Pronase, 0.1 M phosphate, pH 7.5.

Procedure. The flow diagram for the enzyme incubation and analytical system is shown in Fig. 1. The oil-bath temperature was 40°C for the assay of trypsin and Pronase. Aliquots of solutions were sampled at a rate of 40 per hour with a sample to wash ratio of l : l . A sampling pro- gram of 30 per hour with a sample to wash ratio of 1:2 may also be used, which gives greater separation of peaks but also has no influence on the final values.

The dimethyl casein always has a residual low TNBS reaction and this must be used as a base value. Therefore, substrate and analytical reagents are first run through the system. The absorbance at 420 nm should be less than 0.10 and, with carefully prepared substrate, is close to 0.050. Suitable dilutions of enzyme are them sampled. If the protease is of low specific activity, the activity peaks will be increased because of unmethylated amino groups in the enzyme extract. This can readily be corrected for by running enzyme samples again, but with the substrate solution replaced by buffer alone. The total time from enzyme and sub- strate mixing to entrance of incubated sample into the colorimeter flow cell is 45 rain. This includes a residence time for enzyme incuba- tion in the oil bath of 23 min and a residence time in the 40°C water bath for colour development of 6 min. Activity is expressed as glycine equiva- lents released per hour. After use the system is washed with 0.1% (w/v) sodium dodecyl sulfate solution and then with distilled water.

Direct Determination of Amino Nitrogen

Reagents. The reagents are the same as used for assay of proteolytic enzymes, except no substrate is required.

Procedure. The mainfold is a modification of that shown in Fig. 1. The

PROTEINASE AND AMINO NITROGEN ASSAY 183

Oil Bath

U WoOer Both 4.0 °

/VVV

TUBE DIAM 0 0St

I 0 045

I 0 025

Analytical 0 040

Substrate

Air

~ W a ~ t e =. I --

0 065 Air

0 07:5 Borate

0 Of 5 TNBS

0 051 Phosphate Sulphite

Waste 0.08( .]

Waste

F]6. I. Flow diagram for the automated determination of proteolytic enzymes. Standard Technicon equipment was used in the construction of the analytical system.

sampler is merely connected directly to the tube designated" Analytical ." This bypasses the enzyme incubation stage. Aliquots of solution were sampled at the rate of 30 per hour with a sample to wash ratio of 1:2. A sampling program of 40 per hour with a sample to wash ratio of 1:1 is also suitable. Total analytical time is 11 min. 20 sec with a resi- dence time in the 40°C water bath of 6 rain.

RESULTS

Determination of Proteolytic Activity

A copy of the recorder chart trace with an assay of varying concen- trations of trypsin is shown in Fig. 2. The initial base level due to the substrate is first determined at 0.050 units and then the enzyme samples are assayed. After the original baseline is reestablished, the blank due to enzyme alone is determined. The procedure generates classical hyper- bolic curves when substrate concentration is varied (Fig. 3).

184 ADAMS, ROBBERTS AND BUTLER

09 Oa

07

06

05

04

r~

Fxo. 2. Recorder chart trace for assay of varying concentrations of trypsin. Instrument baseline is first established at 0.001 absorbance units, then dimethyl casein baseline at 0.05 absorbance units. Trypsin concentrations of 0.25, 0.50, 1.0, 5.0, 10.0, 15.0, and 20.0 ixg/ml were then sampled. After sampling, the dimethyl casein baseline is reestab- lished followed by instrument baseline. The enzyme solutions are sampled once again without substrate to determine enzyme blank values.

Determination of Amino Nitrogen

T h e a s s a y is l inear in the range o f 2 5 - 2 5 0 nmole s /ml o f a m i n o ac id . G l y c i n e a t 250 n m o l e s / m l g ives an a b s o r b a n c e o f 1.00, l euc ine g ives 0.94, and a lan ine 0.92.

T h e r e s p o n s e o f va r ious sal ts in the ana ly t i ca l s y s t e m w e r e t e s t ed . S o d i u m ch lo r ide , s o d i u m ace t a t e , c a l c ium ch lo r ide , sod ium sulfa te , sod ium p h o s p h a t e , s o d i u m n i t ra te , and p o t a s s i u m ch lo r ide , w e r e w i thou t e f fec t a t a c o n c e n t r a t i o n o f 1 mM. C o p p e r , h o w e v e r , was a s eve re inh ib i to r , g iv ing a r e d u c e d a b s o r b a n c e at a c o n c e n t r a t i o n o f 0.2 mM.

4 0

~ o

zk

0 ~ 2 4 5

DEmelhyl Coseln (mg/ml~)

FIG. 3. Effect of varying dimethyl casein concentration against different levels of Pronase. Pronase concentrations (tzg/ml): ((3) 1.0; (~) 2.5; (Fq) 5.0; (O) 10.0; (A) 15.0.

P R O T E I N A S E A N D A M I N O N I T R O G E N ASSAY 185

Ammonium chloride on the other hand increased the absorbance. The influence of various organic compounds is shown in Table 1. Tri- chloroacetic acid decreases the absorbance while mercaptoethanol, Tris, and ethanol increase the absorbance.

DISCUSSION

The protease system described here is more desirable than many other procedures on theoretical grounds. Activity is measured as an in- crease in free amino groups which are released as a result of proteolysis. This is a more general method than those based on absorbance of peptides at 280 rim. The method is somewhat simpler than others, in that no filtration step (7,8) is required. Furthermore, the reaction is carried out in a homogeneous system. The procedure is particularly suited to the assay of multiple fractions obtained from column chromatog- raphy. Dimethyl casein has similar solubility characteristics to casein and precipitates below pH 5.5 (4). However, if assays are required to be run at a lower pH then other dimethylated proteins, such as dimethyl hemoglobin, may be used.

The amino nitrogen assay is sensitive, rapid, and simple. Unlike the automated ninhydrin procedure, there is no requirement for organic sol- vents nor a nitrogen gas supply (6). The sensitivity is similar to that of the manual method described by Fields (5), and small quantities of samples only are needed for analysis.

Many common inorganic reagents have little effect on the TNBS reac- tion and if the standards reflect the buffer composition or other com- pounds in the samples, then little difficulty occurs. Materials which have significant effect such as trichloroacetic acid and ethanol can also be accomodated if the wash water is replaced by appropriate concentra- tions of these materials. We routinely assay samples for amino nitrogen in 75% (v/v) ethanol or 5% (w/v) trichloroacetic acid. The reduced absorbance due to trichloroacetic acid appears to be a pH effect and

TABLE 1

THE INFLUENCE OF VARIOUS ORGANIC COMPOUNDS ON THE ABSORBANCE OF A STANDARD GLYCINE SOLUTION ASSAYED WITH TNBS a

Organic compound Concentration Absorbance at 420 nm

None 0 0.230 Trichloroacetic acid 2% (w/v) 0.187 Mercaptoethanol 0.05 M 0.990 Tris 0.02 M 0.500 Ethanol 50% (v/v) 0.391

a Standard glycine solution was 50 nmoles/ml.

186 ADAMS, ROBBERTS AND BUTLER

may also be overcome by increasing the concentration of the borate buffer to 0.1 M.

As with the ninhydrin reaction, ammonia is reactive in this system and solutions should be kept free from contamination by ammonia. Thiols also have an instantaneous reaction with TNBS under the conditions used here (5) and this must be considered when analyzing complex mixtures.

REFERENCES

1. Okuyama, T., and Satake, K. (1960) J. Biochem. (Japan)47, 454-466. 2. Habeeb, A. F. S. A. (1966)Anal. Biochem. 14, 328-336. 3. Brinkley, F., Leibach, F., and King, N. (1968) Arch. Biochem. Biophys. 128,

397-405. 4. Lin, Y., Means, G. E., and Feeney, R. E. (1969)J. Biol. Chem. 244, 789-793. 5. Fields, R. (1971) Biochem. J. 124, 581-590. 6. White, L. M., and Gauger, M. A. (1967) in Automation in Analytical Chemistry

(Technicon Symposia), Vol. 1, pp. 213-217, Mediad Incorporated, New York. 7. Hazen, G. G., Hause, J. A., and Hubicki, J. A. (1965) Ann, N .Y . Acad. Sci.

131), 761-768. 8. Newman, P. B., and Etherington, D. J. (1974)Anal. Biochem. 57, 347-355.