Spectrophotometric Determinut io n of Trypsin and Trypsin Inhibitors MARVIN 8. RHODES, ROBERT M. HILL, and ROBERT E. FEENEY Department o f Biochemistry and Nutrition, University o f Nebraska, Lincoln, Neb.
b A simple and rapid spectrophotometric method for the determination of trypsin and trypsin inhibitors is based on the determination of acid production by means of changes in absorption of indicators. When a recording spectrophotometer is employed, the enzyme activities may be obtained directly from the slopes of the transmittance-time curves plotted by the recorder. Amounts of trypsin from 1 to 15 y can be determined in a substrate-buffer-indicator solution of 0.01 3M p toluenesulfonylarginine methyl ester, 0.01 M tris(hydroxymethy1)aminomethane (pH 8.2), and 0.01 % rn-nitrophenol.
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D
URING S I U D I L ~ on
egg protclins in thib laboratory, it became dcsirable to develop a simple and rapid assay for trypsin and trypsin inhibitors. Current methods utilized the esterase or amidase activity of trypsin on FJ iithctic esters or amide\ ( 2 , $ 3 7-9). T h r nctivity \vas rlctcrniincti by titrating the acid prodnccd or b y utilizing the difference in ultraviolet absorption spectra between the substratc and its split products. A much bimpler method seemed possible by photonictric determination of the acid produccd through color changes of an acid-base indicator. The current availability of spectrop1iotoiiic.ters equipped n ith automatic rccoitiing devices would makc possible the determination of enzyme activity simply by comparing thc slopes of transniittancctime curves plotted hy the instrunic,nt.
a t the same temperature as that of the cell compartment. Potentiometric titrations were performed using a Beckman Model G pH meter (2, ?', 8). Stirring was accomplished by bubbling nitrogen through the solution. The trypsin was a twice recrystallized product containing approximately 50% magnesium sulfate as purchased from the Xutritional Biochemical Corp., St. Louis, N o . It contained 37.9% trypsin as calculated on a nitrogen content for pure trypsin of 16.13% (6). Standard solutions of trypsin were prepared on the basis of this nitrogen content and were made up daily in 0.002.Y acetic acid. Ovomucoid was prepared by the method of Lineweaver and Murray (5) and activities were reported on a dry basis corrected to a nitrogen content of 13.3% nitrogen ( 5 ) . The soybean trypsin inhibitor, preparation S15314, was purchased froni Worthington Biochcmiea1 Corp., Freehold, S. J. Egg ivhite \\as hlrndrd (10) and diluted in distilled v a t c r ( 2 ) for assay. The substrate, p-toluenesulfonylarginine methyl ester, was synthesized essentially according to the method of Bergmann, Fruton, and Pollok ( 1 ) . The buffer, tris(hydroxymethy1)aminomethane (Sigma 121, lot 125-130, Sigma Cheniical Co., St. Louis, No.), was dissolved in water m d adjusted to pH 8.2 with hydrochloric acid. Indicators used n ere as follows: m-nitrophenol, Eastman White Label, No. 1340; phenol red (phenolsulfonephthalein) Eastman, Lot S o . 11; m-cresol purple, Fisher Scientific Co.. cresol red; brilliant ycllow; and neutral red. The last three indicator< were from Sational Aniline Division, Allied Chemical and Dye Corp.
APPARATUS A N D MATERIALS
EXPERIMENTAL METHODS A N D RESULTS
A Heckman Modrl IIU spcctropliotometer equipped with photomultiplier attachment was used for all spwtrophotometric dctcrminations. I t n a q connected to a Hristol's Dynaniastc.r rccording potentionietcr through a Beckman energy recording adapter, JIodcl 5800. T h r chart spwd \vas 1 inch per minute. Tlic qmtrophotometvr cell compartnicnt \vas also furnishcd rvitli thermospacm through which \\-ater from a tlierniostated bath was circulated. A constant tc,mprrature (37' C.) was thus maintained during all dctcrminationi. An auxiliary constant temperaturr iiatli mnintainc~lall rc.:igcmts
Development of Method. It was nccessary t o consider t h e following interrelated factors t o obtain a linear change in transmittance over the desired range of change in pH : pH optimum of the enzyme and the suitable range oi change in pH. The buffer and its concentration. The concentrations of substrate and enzyme. The indicators and the proper wave lengths and concentrations thereof. The over-all operating conditions, including volumes of reagents, order of addition, reaction time, and the like.
376 *
ANALYTICAL CHEMISTRY
Schwert and con orkers (8) rcportccl that the maximum rate of hydrolysis of p-toluenesulfonylarginine methyl estcr by trypsin a t 25' C. occurred a t a pH range of 7.9 to 8.4. Buffers were examined which should give a n approximately linear change in pH with addition of acid over this range. Substrate concentrations as previously described (0.01 to 0.02M) (R,6) and 001.11 tris buffer, pH 8.2, n-ere found suitahle Indicators were then chosen n hich would give an approviniately linear change in transmittance x i t h change in pH in tlie buffer selected Confirmation of these linear relationships was obtained by potentiometric titration of the buffer nith acid and by measuring the transmittance of the indicator-buffer solution a t several pH values. m-Sitrophenol a t 395 mp and phenol red at 440, 520, or 565 nip n ere the most ,atisfactory of the indicators studied. The wave lengths indicated viere at absorption maxima, n i t h the evception of 520 nip for phenol red. m-Sitrophenol (O.O1co) gave a linear change over a greater range in pH than did phenol rrd and I\ as, therefore, used. The procedure finally adopted for tlic assay of trypsin 11 as as follom. A I-nil. portion of enzyme solution, containing 1 to 15 7,was placed in the cuvette of the spectrophotometer. and 2 nil. of substrate-buffer-indicator qolutiori [0.02Mp-toluenesulfon~largininemrthyl ester, 0.015-Tf tris(hydroxymethy1)aminomethane at pH 8.2, and 0.015yo nL-nitrophenol, respectively] wa5 added. The recording was started 2 to 3 zeconds after this addition. Adequate mixing of solutions was acconipli*hed bv bloiving the substrate-buffer-indicator wlutioii into the cuvette using a fin+tipped pipet. Direct estiniation of (mviiie activities was possihle 1):. eomp:irieon of the slopes of the linear portions of the plotted curves. Trypsin Assays. A wiles of expeiiinents I\ as performed using various concentrations of tiypdin. Figuie 1 pwsents t h e curves obtained in one evpeiiinent a t t h e concentrations of trypsin indicated. In all cascq, the curves w r e linear for at least 60 seconds. Klien the slope. of theqe ciirves i v r e then plotted againyt concentratiolib of trypsin, a linear relationship \\as ob-
% TRANSMITTANCE Figure 1. Reproduction of curves plotted by recorder for trypsin assay using m-nitrophenol at 395 mp Figures on curves represent micrograms of trypsin; slopes determined below dotted line
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INHISITOR, micrograms Figure 2.
Curves for trypsin inhibitor assay
Each concentration of inhibitor assayed in presence of 15 y of trypsin
Table 1.
Comparison of Methods for Determination of Trypsin and Ovomucoid
3Iethod llaterial Added! "/ Trypsin Ovomucoid
Spectro hotometric., C ( fiminiite 0
1.43 2.iO
15
seconds-'
> 1200
4.00
12 15 15
Spot platej
5.50
3.2 6.5 9.7
I ;i 1 ,i 13.0 15 16 2 K e i y h t ratio. ovomucoid
6.67 5.25
4.70 3.25
2.20
1.20
t .16 t o trypsin Seconds required for indicator t o turn from red to yellow Milliliters 0 1 0.laysodium hydroside added per minutc.
153 G4 12 33 21
31 41
58
130 500 1 2
Titration, ml.jniinriteb
x
10-2
