W2A-023 Peptidase activity-0.txt
VISUAL DETECTION OF PEPTIDASE ACTIVITY USING FLUOROGENIC SUBSTRATES IN A MICROTITER PLATE ASSAY

G. Brent Irvine, Michael Ennis, and Carvell H. Williams Division of Biochemistry, School of Biology and Biochemistry, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, United Kingdom

Received August 14, 1989

A simple, inexpensive, and sensitive assay for peptidase activity has been devised. The assay was performed in a microtiter plate and was based on flourogenic peptide substrates, many of which are commercially available. 7-Amino-4-methyl coumarin the flourescent product liberated during an incubation period of between 1 and 16 h, was detected by inspection of the plate under ultraviolet light of wavelength 356 nm. A fluorometer was not required. Using α -chymotrypsin as a model enzyme, with succinyl-L-alanyl-L-analyl-L-prolyl-L-phenylalanine 4-methyl-coumarlyl-7-amide as substrate, it was shown that as little as 4 fmol of enzyme could be detected. The method was non-quantitative and was particularly suited to location of enzyme activity in fractions during a purification procedure. The validity of the assay was demonstrated by detection of activity of a known enzyme, α -chymotrypsin, after its purification by size-exclusion high-performance liquid chromatography. The method was used to locate two forms of aminopeptidase activity, in fractions from size-exclusion chromatography of an extract from reproductive tissue of Helix aspersa, using L-leucine 4-methyl-coumaryl-7-amide as substrate.

Some of the most sensitive methods for assaying proteolytic enzymes are based on the liberation of a fluorescent product from a fluorogenic reactant. These methods are normally used in a quantitative manner to titrate the enzyme (1) or to estimate its concentration by relating an initial velocity to one given by a standard solution of enzyme (2). A spectrofluorometer is needed for accurate determinations of fluorescent product concentration and the reaction is carried out in a cuvette, a relatively laborious procedure if many samples are to be assayed. Dresden et al. (3) have outlined the savings that accrue in time and reagent volumes when enzyme assays are performed in microtiter plates containing 96 wells. These authors developed a microtiter plate assay for a cathepsin B-like enzyme from Schistosoma mansoni using the fluorogenic substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine 4-methyl-coumaryl-7-amide. The fluorescent product, (AMC), 7-amino-4-methyl coumarin was measured using an inverted fluorescence microscope, enabling the reaction kinetics of the system to be determined. The authors also pointed out the potential of the method for screening for peptidases of different specificity using appropriate fluorogenic peptide substrates. However, in many cases it is not necessary to estimate the concentration of an enzyme or to determine its kinetic behavior but merely to establish whether it is present in a sample. This would apply particularly to the location of enzyme activity in fractions obtained during a purification procedure. It could also be of value in screening potential inhibitors. To this end we have developed a microtiter plate assay, using fluorogenic substrates, that enables us to locate peptidase activity by inspection of the plate under an ultraviolet lamp. No expensive spectrofluorometric instrumentation is needed and a permanent record is easily obtained by photographing the illuminated microtiter plate. The method is illustrated with reference to the purification of aminopeptidase activities from reproductive tissue of the garden snail, Helix aspersa.

MATERIALS AND METHODS

The following enzymes were obtained from Sigma Chemical Co. (St. Louis, MO): α -chymotrypsin Type II (C4129); α -chymotrypsin Type VI inactivated with DEP (C1012); leucine aminopeptidase (LAP) Type III-CP, from porcine kidney cytosol (L9876). Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-methyl-coumaryl-7-amide (Suc-AAPF-AMC) was purchased from Peninsula Laboratories, Inc. Belmont, CA). Succinyl-L-analyl-L-phenylalanine 4-methyl-coumaryl-7-amide (Suc-AAF-AMC) was supplied by Sigma. AMC was prepared by the method of Zimmerman et al (2). L-Leucine 4-methyl-coumaryl-7-amide (L-AMC) was prepared by the general method for the preparation of amides described by Yasutake and Powers (4).

Absolute concentrations of solutions of α -chymotrypsin were determined by titration with 4-methylumbelliferyl-p-trimethylammonium cinnamate chloride (Sigma) using the method of Jameson et al. (1) and a TKO 100 minifluorometer (Hoefer Scientific Instruments, Newcastle, UK). Microtiter plate assays for peptidase activity were carried out in a MircoFLUOR plate (Dynatech, Billinghurst, UK) covered with a plastic lid. A stock solution (8.1 mM) of Suc-AAPF-AMC was made up in dimethyl sulfoxide. All other solutions were made up in 0,1 M Tris-HCI buffer, pH 7.4, containing 10 mM CaCl 2. Each well contained a final volume of 375 μ l. To view the plate it was placed between two dual-wavelength ultraviolet lamps (Baird and Tatlock, Romford, UK) operated at 356 nm. The photograph shown in Fig. 1 was taken using an Olympus OM-1 camera equipped with a Zuiko MC Auto-Macro lens fitted with uv (0) and polarizing filters (Hoya). Kodak Tri-X 135 film was exposed for 10 s at aperture f 11.

Reproductive tissue was dissected from freshly killed snails and was frozen in liquid nitrogen. On partial thawing, the tissue was finely chopped using a scalpel and was then homogenized in 0.1 M Tris-HCI buffer, pH 7.4, containing 1 mM CaCl 2, 1 mM dithiothreitol, and 0.25 M sucrose (1 g tissue/4 ml buffer) at 4 ̊ C. The homogenate was centrifuged at 50,000g for 30 min at 4 ̊ C and the supernatant was stored at -20 ̊ C. The protein concentration of the supernatant was measured by the method of Lowry et al. (5) using bovine serum albumin (Sigma A4378) as standard.

High-performance size-exclusion liquid chromatography of this supernatant was carried out in 20 mM Tris-HCI buffer, pH 7.0, containing 0.25 M NaCl and 10 mM CaCl 2 on a TSK preColumn (7,5 X 75 mm) linked in series with two TSK-G2000-SW columns (7,5 X 300 mm) from LKB (Uppsala, Sweden)(Fig. 2). The chromatography was performed with a Model 510 pump, a U6K injector, and a 441 absorbance detector (Waters, Harrow, UK) linked to a 2001 series chart recorder (Vitatron, Maidenhead, UK). Fractions (0.5 ml) were collected manually and were stored at -20 ̊ C prior to assay.

RESULTS AND DISCUSSION

Incubation of peptidases for 16 h at room temperature (about 20 ̊ C) with an appropriate fluorogenic substrate produced a fluorescent product that was visible upon illumination of the microtiter plate with ultraviolet light (Fig. 1). Incubation times of a few hours were normally sufficient and an overnight incubation was only needed in cases where enzyme activity was very low. For example, in the assay shown in Fig. 1, activity was detectable after a 3-h incubation in all the wells that became strongly fluorescent after 16 h.

Whereas AMC has excitation and emission maxima at 345 and 445 nm, respectively, when the amino group is converted to an amide, for example in a fluorogenic peptide substrate, these values are shifted down to 325 and 395 nm, respectively (2). Commercially available ultraviolet lamps emit light of 356 nm, which corresponds closely to the desired excitation maximum for AMC. Under such illumination, fluorogenic substrate of concentration 10 μ M emitted a dark purple fluorescent light that appeared to the naked eye to be much less intense than that due to an equivalent concentration of AMC. There was, however, a definite background fluorescence in substrate-containing wells. This can be seen by comparison of well B10 (Fig. 1), containing 10 μ M Suc-AAPF-AMC, with well A10, containing only buffer. This background increased markedly if concentrations of substrate in excess of 10 μ M were used, making it more difficult to differentiate between wells where AMC was generated and those where it was not. Nonetheless, it was possible to use substrate concentrations up to 100 μ M since the intensity of the fluorescence due to the higher concentration of AMC produced under these conditions compensated to some degree. This would be particularly useful for substrates with relatively high K m values for the enzyme, where the reaction might otherwise be very slow. Because the Tri-X film used for the photograph in Fig. 1 was sensitive to ultraviolet light, a uv(0) filter with a cut-off value of 400 nm was used to give a photograph that more accurately matched the image seen with the naked eye. A polarizing filter was used to minimize light reflected from the glossy black surface of the MicroFLUOR plate. This plate is designed for assays based on fluorescence and is made from opaque black plastic so that light is confined within each well and does not illuminate adjacent wells, as happens with transparent plates.

Standards containing decreasing concentrations of AMC were added to wells 1 to 10 in Row A. Concentrations below 1 μ M (wells A7-A10) did not produce visible fluorescence. The use of substrate concentrations of 10 μ M appeared to be optimal in terms of minimizing intrinsic fluorescence due to substrate yet providing sufficient product to see clearly. It also conserved substrate. For example, 1 mg of Suc-AAPF-AMC was sufficient for 400 wells. The visual assay was intrinsically less sensitive than a spectrofluorometric assay, since concentrations of AMC 100-fold lower could be measured using an instrument. Nevertheless, very low concentrations of peptidase activity could easily be detected after an overnight incubations. From Row B it is clear that a concentration of α -chymotrypsin of 0.2 ng/well (B5) gave rise to clear fluorescence after 16 h at 20 ̊ C. The well B1 became strongly fluorescent immediately after addition of enzyme (0.2 μ g protein/well) after a few minutes, and B3 (20 ng protein/well) within 30 min. The presence of other proteins did not affect the method, since inclusion of 0.01% serum albumin in all solutions did not alter the results illustrated in Row B (data not shown). The results shown in Row C were obtained using the same conditions as those in Row B except that α -chymotrypsin that had been inactivated with DFP was used. This material was stated to have residual activity of 0.1 unit/mg compared to 52 units/mg for the uninhibited enzyme. This 500-fold decrease in activity was clear in our assay since only at a 1000-fold higher concentration (0.2 μ g protein/well, well C2) was a positive result obtained. The operational molarity of the stock solution (0.1 mg/ml) of α -chymotrypsin, determined by titration with 4-methylumbelliferyl-p-trimethylammonium cinnamate chloride, was found to be 2.084 ± 0.004 μ M (mean ± SD of six measurements). This compared with a theoretical value of 4.27 μ M based on weight, so that the lyophilized protein contained 49% active enzyme. Consequently, the amount a α -chymotrypsin present in well B5 (the lowest concentration of enzyme that gave a positive result) was actually about 0.1 ng or 4 fmol. Thus, using an overnight incubation it was possible to detect very low concentrations of enzyme, provided that a good substrate (one that was substantially converted to product during this period) such as Suc-AAPF-AMC was available. On the other hand the substrate Suc-AAF-AMC did not provide as sensitive an assay for α -chymotrypsin and concentrations of the enzyme two orders of magnitude higher than those detectable using Suc-AAPF-AMC were needed to give a positive result (data not shown). L-AMC did not provide a very sensitive assay for LAP. Only at concentrations of at least 3.8 μ g protein/well (well D2) was LAP detectable using L-AMC as substrate. The sensitivity was increased 100-fold, however, if calcium in the assay buffer was replaced by magnesium, which has been shown to activate LAP (6). Thus, in the presence of MgSO 4 (5mM), LAP at a concentration of 38 ng protein/well gave a weakly positive result (data not shown).

The method has been applied to the detection of peptidase activity in fractions obtained during a purification procedure. Two aminopeptidase activities, separated by size-exclusion chromatography of a preparation from reproductive tissue of H. aspersa (Fig. 2), were readily detected (wells E1-E4 and E7-F1). Indeed wells E2 and E8, where maximal activities were found, gave positive results after incubation for 30 min. The apparent molecular weights of proteins eluted in fractions 2 and 8, corresponding to the peak position of the two iminopeptidase activities, were estimated, by comparison with the elution volumes of immunoglobulin G and serum albumin, to be 160,000 and about 65,000, respectively.

In order to establish that under ideal conditions the microtiter plate assay detects the appropriate material, a sample of a known enzyme, α -chymotrypsin, was subjected to size-exclusion chromatography on the same system. From inspection of Fig. 3 it can be seen that the peak of enzyme activity in fractions 19 and 20 corresponded exactly to the protein peak as measured by absorbance at 214 nm.
