One Step Protease Assay Technical Brief
G. P. Royer1 and Sheldon E. Broedel, Jr.2
Buford Biomedical, Frederick, MD1
Athena Environmental Sciences, Inc., Baltimore, MD2
We have developed a general protease substrate consisting of cross-linked albumin,
azoalbumin, and gelatin. The substrate is susceptible to proteolysis by a wide range of
enzymes including: collagenase, papain, bromelain, trypsin, chymotrypsin, proteinase K,
and pronase. The assay can be run in screw-cap vials or multi-well plates for high
throughput screens. Crude enzyme preparations which contain particulate matter can be
assayed using a back-digestion technique for measurement of residual solid. Nucleophiles,
such as thiols, in the assay buffers do not interfere. The limit of detection of enzymes
is less than 100 ng for most enzymes tested. The very low labor input and "hands-on"
attention are decided advantages.
Interest in proteases has increased with the realization that they play key roles
in rheumatoid arthritis1 and cancer metastasis.2,3 Tumor progression depends on
remodeling of basement membrane prior to invasion and angiogenesis. Some inhibitors of
the proteases which catalyze these processes have shown good anti-tumor activity without
the side effects of cytotoxic drugs.
In addition to those investigators who target proteases for research, biochemists
are generally concerned with protection of their valuable proteins from unwanted degradation
by contaminating proteases. These workers need to verify the presence or absence of proteases
in their preparations.
Modern methods have supplanted the classical three-step assay-digestion, TCA
precipitation, and detection of TCA soluble peptides by UV absorbance, the Folin-Ciocalteu
reagent, or other means. Two currently popular assays are based on derivitized casein.
In 1984, Twining introduced a widely applicable and sensitive assay based on fluorescein
isocyanate-labelled casein.4 This assay procedure suffers from the cost of the substrate
and labor intensity.
In 1992, Hatakeyama, et al., reported an assay based on succinyl-casein and the
TNBS reagent for detection of liberated amino groups.5 This assay can be run with the
multi-well plate reader. Disadvantages include failure to detect collagenases, background
due to reactive amines or thiols, and interference from particulates in the sample.
Our goal was to formulate a pipettable liquid which could be conveniently dispensed
into microplates or vials. A number of cross-linking reagents and numerous sets of reaction
conditions were tested before we settled on the procedure described here which employs
formaldehyde, concentrated protein solutions and sodium benzoate.
The substrate described here is a mixture of gelatin and albumin cross-linked in
the presence of sulfaniloazo-albumin with formaldehyde at slightly acidic pH. The translucent
solid is mechanically stable even at elevated temperature. Twenty-four hour backgrounds
without protease are acceptably low, when the assay mixture contains 0.1 % azide. Collagenase,
papain, bromelain, trypsin, chymotrypsin, proteinase K, and pronase all exhibit good activity
against this protein matrix.
Materials and Methods
All chemicals were reagent grade or better. Proteins and enzymes were supplied by Sigma
as follows: albumin (A2153), sulfanyloazo albumin (A2382), gelatin (G2500), collagenase C0773,
papain (P4762), trypsin (T7409), chymotrypsin (C7762), proteinase K (P5568), and pronase (P0652);
partially soluble enzyme preparations pancreatin (1x, 4x, and 8x) and bromelain (B22520), were
used as supplied. Papaya latex (P3375) was ground to a powder before use. Assay buffers and
storage solutions were prepared as previously described (6).
Vial Assays. In a typical assay, 500 µl of reaction buffer and 100?l of enzyme
solution were added to vials containing the substrate matrix, the vials sealed tightly, and
incubated at 37°C. The duration of the incubation was varied from 5 min. to 24 hours.
To stop the reaction, 500 µl of 0.2N NaOH was added to each vial. The absorbance at 450nm
of the aqueous phase was measured spectrophotometrically.
For enzyme samples containing particulates, such as the crude preparations studied
here, a back digestion technique was employed. After reacting the crude preparation in the
vial assay, the assay mixture was poured off and the solid substrate washed with water at
the end of the reaction period. The extent of hydrolysis was established by digesting the
remaining substrate with an excess of enzyme such as proteinase K (100µg). The amount of
substrate digested by the crude material was determined by the difference between the no
protease control (which represents the total amount of substrate) and the experimental
(the residual amount of substrate). This number divided by the control value and multiplied
by 100 equals the percent maximal hydrolysis.
Multi-well Plate Assays.
Alternate columns of 96-well plates (Corning 25880-96) were filled with substrate. The
remaining empty wells were used as working wells for absorbance measurements. A typical
assay consisted of 100 µl reaction buffer containing the respective protease. After the
elapsed reaction time at 37°C, 50µl aliquots were transferred from the assay wells
to the respective adjacent working wells. To each working well 50µl of 0.1 N NaOH was
added and the absorbance was read at 450nm with a microplate reader (Molecular Devices,
Results and Discussion
General characteristics of the protein-gel matrix were assessed by examining matrix
deterioration under different environmental conditions. The matrix was found to exhibit
good thermal stability, pH stability, resistance to microbial attach, and susceptibility
to a wide variety of proteases including collagenase, papain, bromelain, trypsin, chymotrypsin,
proteinase K, and pronase. This list encompasses a diverse range with regard to active
site structure and mechanism.
Figure 1 shows time courses of the hydrolysis catalyzed by collagenase,
proteinase K, papain, and chymotrypsin in the vial assay. All plots were linear for
the first two hours, and within 6 h, all of the substrate was consumed. The rates are
significantly higher for collagenase (25 µg) and proteinase K (25 µg) than for papain
and chymotrypsin (50 µg of both in assay). Reactions containing lesser amounts of
proteases (down to 50 ng) also showed linear rates but required up to 20 h incubation.
Similar results were obtained with the microplate assay (data not shown).
Figure 1. Vial assay showing the time-course of papain, chymotrypsin proteinase K, and
collagenase. The total volume was 600ul and the temperature 37°C. To produce
these curves required 25µg of collagenase and proteinase K; papain and chymotrypsin
were present at 50µg/assay.
Figure 2. Rate versus enzyme concentration for collagenase for both the vial assay and
the microplate assay. The limit of detection was less than 30ng.
To assess microenvironmental effects, we examined the ph-dependencies of
several enzymes. Plots for the chymotrypsin-catalyzed hydrolysis of N-acetyl-tryptophan
amide and the azo-albumin gel versus pH show very similar profiles (Figure 3). These
results suggest the absence of strong microenvironmental effects coming to bear as a
result of diffusional limitations at the interface between the solid substrate and the
Figure 3. pH-dependences of chymotrypsin with N-acetyl-tryptophanamide and the azoalbumin
substrate. The similarity of the ph-profiles would suggest the lack of any strong
diffusional limitations at the interface of the solid substrate and the bulk solution.
One significant advantage of this assay system is the ability to directly
test enzyme samples containing particulates. The enzyme along with suspended material
was incubated with the substrate for the desired time. On completion of the reaction
the vial or microplate was washed with water and the remaining substrate digested with
an excess of any protease. The difference in absorbance of the control and experimental
sample represents the amount of digestion. Five crude enzyme preparations which contained
particulates were tested with this back digestion format. The assays were incubated for
one hour at 37°C with 5mg of crude enzyme. Following incubation, the vials were washed
and the residual substrate exhaustively digested with 100?g proteinase K. The results,
shown in Table 1, were that substrate digestion using particulate containing solutions
of bromelain and pancreatin was measurable by this technique. This demonstrates that
protease activity in crude preparations can be quantified with this matrix assay.
Table 1. Hydrolysis of azoalubumin gel catalyzed by crude enzyme preparations
containing particulate matter.
This assay may be useful as a high throughput screening device for detection
of protease inhibitors such as matrix metalloproteinase inhibitors. To illustrate, we
used the microplate format to study the inhibition of trypsin by pancreatic trypsin
inhibitor. Trypsin (2 µg, 2.24 BAEE units) was mixed with different amounts of bovine
trypsin inhibitor and incubated for 5 min. The residual trypsin activity was measured
in the microplate assay. The results were consistent with known stoichiometry (Figure 4).
Figure 4. Dose-dependent inhibition of trypsin by bovine trypsin inhibitor.
Inhibitor at various concentrations, was incubated with 2µg (2.24 BAEE units) of trypsin
for 5 min. Residual trypsin activity was measured in the microplate protease assay as
described in Material and Methods for 20 h at 37°C.
The assay described here is a rapid, low-cost, easy-to-use means of measuring
protease activity. Through different formats, the assay can be applied to a rapid spot
test (vial assay) for routine protease test during the preparation of biologicals, or
when formatted in a multi-well microplate, applicable to high-throughput screens used
to identify pharmecutically activity compounds. To devise an assay with a specific
minimum cut-off would be straightforward. The reaction time and thickness of the
substrate layer could be set to reflect the desired sensitivity. A microplate well
exhibiting a "hit" or positive fraction would be have solid substrate remaining above
background as judged by direct reading or by back-digestion.
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