Fluorometric BENON From
H. J. BIELSKI
Brookhazvn New York
’ FREED Laboratory.
Because of their aromatic character, characteristic fluorescence is emitted from the well-known specific substrates of cr-chymotrypsin, lYacetyl-L-tryptophan ethyl ester (ATrEE) and N-acetyl-L-tyrosine ethyl ester (ATEE) and their corresponding products, LV-acetyltryptophan (ATr) and N-acetyl-L-tyrosine (AT). The amino acids into which the substrates are hydrolyzed fluoresce several times more strongly than the esters when excited, respectively, at, 290 and 270 mp. The relative fluorescence intensities are given in Table 1. This factor, coupled with the
TABLE INTENSITIES Excitation
1 OF FLUORESCENCE Emission
Relative units of deflection at maximum emission
ATrEE .1Tr ru-Chymotrypsin
290 390 290
365 365 337
1.00 2.83 !I.50
ATEE AT a-Chymotrypsin
270 270 270
307 307 365
1.00 4.76 119.00
The above compounds were studied in HCl-Tris buffer, pH = 7.28, at 25 + O.l”C. The results were obtained for a specific setting of slits on the photometer and a concentration of 1 X 10e6 A4 for all substances. Groups A and B were measured on different scales.
high sensitivity possible in fluorometiy, increased the detectability of changes in substrate concentration to considerable lower levels than can be measured in these substances by conventional methods (1, 2). ’ Research
of the U. S. Atomic 192
C’herrCds. The a-chymotrypsin used in this study was three times crystallized (Worthington Biochemical Corp. 1. Standard solutions were prepared by dissolving known amounts of chynnotrypsin in 1Om”S HCl solution at 0°C. These solutions wcrc filtered, by letting them run by gravity through glass sintercd filters. The conccntrwtions of cu-chymotrypsin in these solutions were detc~rtiiinecl from the olkical densities at 282 IllE” (1). ATrEE, ATr, and ATEE were from Mann Research Laboratories, while AT was A grade from Calbiochem. Before use, the esters were sublimed under reduced pressure, giving the same results as some unsublimed samples, and the free acids were recrystallized from alcol~olwater mixtures. All buffer solutions were prepared from triply distilled water and reagent-grade chemicals, with the exception of the hydrochloric acid, which had to be redistilled and the tris (hydroxymethyl) aminomethane (Sigma Chemical Co. j , which was purified by sublimation. Appalwks. The fluorometer was an assembly of the usual type. The source for excitation was a high-pressure mercury arc (BH-6, 1000 watts, General Electric Co.), two monochromators (500-mm focal length and 3.3 m~/mm dispersion, Bausch & Lomb Optical Co.) at right angles, the first to select the exciting radiation, the second to analyze the emitted fluorescence from the sample at t’he extended intersection of the two monochromators. i2t the exit slit of t’he second monochromator was a photomultiplier (Xo. 62.56 B, EMI/US Ltd., Electron Tube Division, Westbury, N. Y.) whose amplified current was recorded. The slits of the monochromator selecting the excitation radiation were 2 mm wide while those of the monochromator for the emission were 3 mm wide. An electronic device called a “zero offset” permitted t,he intensity of the fluorometric background to be brought to zero on the scale of measurement. The sample tubes were of fused silica and the glassware of Pyrex, all cleaned in boiling mixtures of nitric and sulfuric acids, rinsed finally in triply distilled water, and dried in a clean oven at 120”. Assay. For a given fluorometric assay, the substrate was dissolved in 1 ml of methanol (distilled, 30 plate) and diluted to 100 ml xvith the appropriate buffer solution. The concentrated solution (1 x lOma111) n-as then diluted to the desired concentrations and used for the assay as well as for calibration of the photometer. Similar solutions were prepared for the assay products -4Tr and AT which were used for calibrations. The concentration of substrate and of product or both, should not exceed lo-* ~11and that of the enzyme 5 X lo-‘; 31 if linearity is desired betvveen
concentration and fluorescence intensity. Below this concentration the fluorescence of the amino acids proved to be purely additive with that of Lu-chymotrypsin over the pH range of interest. The assay was carried out by placing 4 ml of substrate solution into a silica cell and injecting 100 h of enzyme solution. The assay was then followed by the change in fluorescence intensity. The hydrolysis of ATrEE could best be followed when excited at 290 mp and measured at, 370 mp, while that of ATEE with excitation at 270 rnp and emission at 307 m/l. The reproducibility for these measurements was of the order of 27C, which was made possible by monitoring the intensity of the light source before and after each rate study. The intensity of the light source was sufficiently stable for several hours at a time. In very dilute solutions where duration of assay is long (20-30 min), slow photochemical decomposition of cneymc by thr exciting radiation became significant unless the solution was shielded from it by a shutter except, of course, in those short intervals requirecl for measurement. All experiments were carried out. at 25 2 O.l”C. DISCUSSION
The fluorescence spectra of a-chymotrypsin and the tryptophan and tyrosine derivatives may be seen in Fig. 1. Xo correction for dependence of reflectivity of grating or of phototube response upon wavclcngth were applied (3)) nor were they necessary in the present relative measurements. As calibration, the fluorescence of known concentrations of all the pertinent substances wa,q measured under the conditions of assay. The fluorescence of the substrates and the amino acids changes little in the pH range favorable for assay of oc-chymot,rypsin: pH 7-8.5. The dependence on pH of the various fluorescence intensities at the spectral maxima is indicated in Fig. 2. Only below pH 5.5 does the intensity of the fluorescence of ATr diminish with further reduction in pH, and below pH 6.5 that of AT falls t,oward the intensity emitted by t,he ester. As may be seen in Fig. 2, the fluorescence of the enzyme exhibits a gent.le maximum between pH 7 and 8.5. The wavelength of its spectral maximum shifts from 337 rnp at pH 3.4 to 348 rnp at. pH 9.45; those of t,he esters and amino acids remain unchanged. Fluorescence yielded the same value for the rate of enzymic hydrolysis as did differential absorption spectra in the range of concentrations where both methods were applicable (see Fig. 3). In solutions too dilute for assay by absorption spectra, fluorescence continued to give the same linear relation as at the higher concentrations (Figs. 3 and 4). When the substrate ATrEE was lO-‘M. the lowest concentration of a-chymotrypsin detected as catalyst was 10.I1 M. While the limit in rate detection was controlled by the product of concentrations of (enzyme) (sub-
FIG. 1. Emission spectra of (1) (3) cy-chymotrypsin 1.00 X 10d M 290 mp. Emission spectra of (4) Tris-HCl buffer, pH = 7.28 when
strate) as low tration. in Fig. The
3800 Wave length
ATr 1.0 x W&f, (2) ATrEE 1.0 x 1Oj M, and in Tris-HCl buffer, pH = 7.28 when excited with AT 1.0 x 10d M and (5) ATEE 1.0 x l(r5 M in excited with 270 mp.
>1&15 M*, ATr by itself could be detected in concentrations as 10-l” M and the enzyme, by itself at a somewhat lower concenThe results of a study of pH effect on assay rate are summarized 5. sensitivity of the fluorescence method was especially marked at
--- 7- --.~:-.r-\-j ENZYME
FIG. 2. Change in fluorescence intensity with pH: (a) cu-Chymotrypsin, ATrEE, and ATr were excited at 290 mp and emission was measured at 337 and 365 rnh respectively. (b) ATEE and AT were excited at 27’0 rnp and emission intensity was measured at 307 rn.u (the maximum). The concentrations of all compounds were 5.0 x lo-'M except for the enzyme, which was 3.34 x lo-’ M. (c) The following buffers were used: pH 3.4-5.6 acetate; pH 6.3 phosphate; pH 7.3-9.45 Tris-HCl. I
II II llllll llllll
FIG. 3. Change in rate of assay with change in concentration of substrate (ATrEE) at 25°C in Tris-HCI buffer, pH=8.0, ionic strength 0.05. Concentrations of (Ychymotrypsin 3.0 x 10-‘&I. (a) Assay by fluorometry. (0) Assay by differential absorption spectra. 196
I I I IllI
I II 1111
I I I IllI
10-g MOLES /LITER
I lllll~ 10-7
Fro. 4. Rate of assay of wchymotrypsin as a function of its concentration at 25°C. Concentration of substrate (ATrEE) 5.0 x lad M in Tris-HCI buffer, pH 8.16, ionic strength 0.05.
low concentrations of substrate and of enzyme. These experiments will not be discussed here except to mention that the ester hydrolysis of ATrEE at lO-* M could be observed when the chymotrypsin was 10WTM. SUMMARY
The enzymic hydrolysis by cY-chymotrypsin of the substrates, Nacetyl-n-tryptophan ethyl ester and N-acetyl-n-tyrosine ethyl ester, was followed by means of fluorescence whose intensity increased threefold and fourfold per mole, respectively, as substrate was transformed into amino acid. The assay by fluorescence was several orders of magnitude more sensitive than the assay by differential absorption spectra of these substances and was in agreement with it in those concentration regions where both methods overlap. To maintain linearity between concentration and fluorescence intensity, the concentration of substrate should be
FIQ. 5. Change in rate of assay with pH: (1) hydrolysis of ATrEE; (2) hydrolysis of ATEE. The concentration of both substrates was 1 X lv M, while that of the enzyme was 5 x 10-s M. Ionic strength 0.050, temperature 25 + O.l”C. The following buffers were used: pH 46-5.6 acetate; pH = 6.9-7.0 phosphate; pH 7.3-9.45 Tris-HCl.
no greater than 1C4M. In such solutions the rate of ester hydrolysis could be followed with the enzyme at 1CFM. ACKNOWLEDGMENT We wish to acknowledge
with thanks the cooperation
of Mary H. Vise.
REFERENCES 1. SCHWERT,
G. W., AND TAKENAKA, Y., Biochim. Biophys. Acta 16, 570 (1955). 2. CUNNINQEAM, L. W., AND BROWN, C. S., J. Biol. Chem. 221, 267 (1956). 3. UDENFRIEND, S., “Fluorescence Assay in Biology and Medicine.” Academic Press,
New York, 1962.