A sensitive assay for proteolytic enzymes using bacterial luciferase as a substrate

ANALYTICAL

61, 280-287

BIOCHEMISTRY

A Sensitive

Assay

(1974)

for Proteolytic

Luciferase DAVID

NJUS,” The

Received

THOMAS

Enzymes

using

Bacterial

as a Substrate’ 0. BALDWIN,3

J. W. HASTINGS

AND

Biological

Laboratories, Harvard University, Cambridge, Massachusetts 02138 February

27, 1974;

accepted

April

3, 1974

A general assay for proteolytic enzymes using bacterial luciferase as a substrate is described. This luciferase is rapidly inactivated by unusually small amounts of different proteases having a wide spectrum of specificities. The activity is lost exponentially; the pseudo-first-order rate constant for inactivation is proportional to the amount of protease. Since luciferase activity can be measured by a very simple and rapid assay, it affords an accurate, sensitive, and convenient assay for proteolytic activity. This technique is capable of detecting as little as 20 ng of trypsin, requires no centrifugation, and is not hampered by the presence of contaminating pigments in the protease preparation. It is compared at pH 6, 7, and 8 to the phenol color assay and the azocoll assay using seven different proteases.

In studies concerned with elucidating the structure and function of bacterial luciferase it was discovered that small amounts of protease cause a rapid pseudo-first-order loss of activity (1). Since the luciferase assay is simple, rapid, and sensitive (2), it seemed that its inactivation might provide a convenient assay for proteases in general. We therefore tested seven proteases at three different pH values and found that bacterial luciferase does indeed provide an excelIent general substrate. The results are presented with a comparison to two spectrophotometric assays: the azocoll assay (3)) and the phenol color assay (4). METHODS’

Bacterial luciferase was obtained from Sigma and compared with bacterial luciferase (approx 5% pure) prepared according to the method of Gunsalus-Miguel et al. (5) with the addition of a final step involving filtration on a Sephadex G-200 column. Trypsin and chymotrypsin were ‘This work was supported in part by Grants tion (GB 31977X) and the National Institutes ’ N.I.H. Predoctoral Trainee in Biophysics. ’ N.I.H. Postdoctoral Fellow GM 52918. Copyright All rights

@ 1974 by Academic Press, of reproduction in any form

280 Inc. reserved.

from the of Health

National Science (GM 19536).

Founda

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281

ASSAY

obtained from Worthington; thermolysin and aeocoll were obtained from Calbiochem. The other proteases, casein, and firefly luciferase were obtained from Sigma. All proteases were used without further purification. Bacterial luciferase (100 pg/ml) was treated with protease in a volume of 100 ~1 at 25”C, in 0.05 M phosphate buffer, 10U4M DTT” at pH 6, 7, or 8. Proteolysis was stopped by dilution of a lo-,~l aliquot into 1 ml of the reaction mixture used for the luciferase assay (0.02 M phosphate, 0.2% BSA, 1CV M decylaldehyde, pH 7). The bioluminescence reaction was then initiated by injecting 1 ml of 5 X lo-” of FMNH,; the activity was measured in a photometer (6). The azocoll assay (3) was performed with 4 mg azocoll suspended in 1 ml 0.1 M phosphate buffer. An aliquot of protease (5, 20, or 50 ygl was added and the mixture incubated at 25°C. After 10 min, the azocoll was removed by centrifugation and the absorbance was read at 520 nm. The phenol color assay (4) was performed with heat denatured casein. For each assay, 0.5 ml 1% casein in 0.1 M phosphate buffer was mixed with 5, 20, or 50 pg of protease and enough H,O to bring the final volume to 0.6 ml. After incubation for 10 min at 25”C, 1 ml of 1 x TCA was added. The precipitate was removed by centrifugation. To 0.5 ml of the supernatant, 1 ml 0.5 M NaOH and 0.3 ml phenol reagent (Fisher 2 N solution diluted threefold) was added, the sample incubated at 25°C for 10 min, and the absorbance read at 750 nm. Absorbances were measured on a Cary model 15 spectrophotometer. RESULTS

The time course for the loss of luciferase activity in the presence of seven different proteases is shown in Fig. 1. The pseudo-first-order rate for the exponential decrease in activity was found to be dire&y proportional to protease concentration, as shown for trypsin in Fig. 2. The protease assay is quite reproducible using different batches of luciferase. An experiment similar to that shown in Fig. 2, but done 10 mo earlier, gave an apparent second-order rate constant5 for inactivation of luciferase of 10.4 mleng trypsin-l.min-’ compared to the value of 9.4 ml*ng trypsin-l*rnin-l shown in Fig. 2. Most, of the difference is probably due to deterioration of the specific activity of the trypsin rather than to error in the assay. For very low concent.rations of proteases (Fig. 3), it is necessary to continue the incubation for a longer period of time (+l hr), and to run ‘Abbreviations FMNH,, reduced ‘This is not dependent upon

used are: DTT, flavin mononucleotide; a true second-order the initial luciferwe

dithiothreitol; BSA, bovine serum TCA, trichloroacetic acid. rate constant because the observed concentration: see discussion.

albumin; rate

is

282

NJUS,

BALDWIN

AND

HASTINGS

and

t I

2

Time

I

I

3

4

Bromeloin

I 5

I 6

(A

I 7

(minutes)

FIG. 1. Inactivation of luciferase as a function of time of incubation with seven proteolytic enzymes. Bacterial luciferase (0.046 mg/ml) was incubated with proteases (50 pg/ml) in a volume of 100 ~1 0.05~ PO1 buffer, pH 7.0 at 25°C. At the times indicated, 10-J samples were withdrawn and assayed for luciferase activity as described in Methods. The straight lines for the exponential decay represent least squares fits to the data points.

a control to correct for changes in activity

in the absence of protease. Four controls and four incubations with 500 rig/ml of trypsin show that there is a clear decrease in the presence of trypsin, compared to a slight increase in the controls (possibly due to a slight increase in room temperature during the time of incubation). The differences are clearly significant and suggest that the assay should be usable down to 200 r&ml trypsin, or in a volume of 0.1 ml (as used here), 20 ng trypsin. Correcting for the controls, the apparent second-order rate constant for inactivation of luciferase ( ~100 yg/ml) is 11.4 ml*ng trypsin-‘-min-’ compared to a value of 9.4 mleng trypsinP*min-l obtained at higher trypsin concentrations (Fig. 2). As shown in Fig. 4, this assay is applicable to a wide range of proteases; seven were selected and tested at three pH values (6, 7, and 8), and compared to both the azocoll assay (3) and the phenol color assay (4). The results are shown (Fig. 4) with the luciferase assay expressed as the pseudo-first-order rate of inactivation and the other two assays as solubilized absorbance. The luciferase assay is consistent with the

A BIOLUMINESCENT

Trypsin

PROTEASE

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283

(&ml)

FIG. 2. The relationship between trypsin concentration and the pseudo-first-order rate constant for the inactivation of luciferase. A stock solution of trypsin (1 mg/ml) was made up and diluted as needed to give the concentrations tested. To determine each rate constant, lucifersse (0.048 mg/ml) was incubated with trypsin in 0.1 M PO, buffer, pH 7.0 at 25”C, and assayed as a function of time as in Fig. 1. At each trypsin concentration, four rate constants were obtained and their average and standard deviation are plotted. These average first-order rate constants for inactivation have been fitted by the least squares method to give the line whose slope is 9.4 mleng trypsin?*min-‘.

I

I

I

20 40 Time (minutes)

I

I

60

FIG. 3. Assay of trypsin at a concentration of 500 rig/ml; 100 pl luciferase (0.097 mg/ml) in 0.1 M PO,, pH 7.1 was incubated at 25” with and without trypsin; lo+1 samples were withdrawn at lo-min intervals and assayed for luciferase activity. Four samples with 500 rig/ml trypsin have an average inactivation constant of 0.0036 * 0.0014 min-‘. Four control samples have an average inactivation constant of -0.0021 -C 0.0008 min-‘. The average and standard deviation of the four samples at each assay time are plotted. Also shown are the least squares straight lines fit to these average values.

284

NJUS,

6 ?

6

7

8

6

78

AND

CH YMO7RYPSIN

t

7

8

Casein

6

Luciferose

Luciferase

BALDWIN

7

678

8

6

6

7 Casein

6

I I

7 Azocol

0

SU6’TILfSIN

6

Azocoll

Cosein

HASTINGS

8 I

7 Azocol

678

6

7

6

7

Luciferase

6

Casein

8

6

Luciferase

8 I

78

Luciferase

7

Azocoll

8

6

Casein

0

6

7 Cosein

7 Azocol

8

6

7

8 I

0

Azccoll

FIG. 4. Comparison of protease assays. The luciferase assay graphed as pseudoto two speetrophotofirst-order rate constant for inactivation in mine1 1s . compared metric assays graphed as absorbances. The assays were performed at three pHs (6, 7, and 8) as described in Methods. Casein was used as the substrate in the phenol color assay. Protease concentrations in &g/ml are indicated on each bar. Background assays (no protease added) are shown in black.

others with regard to the relative activities it assigns to the different proteases. Furthermore, the apparent pH dependences of the proteases are similar in all three assays, despite the fact that the luciferas? assay presumably depends upon the conformation of luciferase which itself might be pH dependent. Actually, the proteolytic activities as assayed by luciferase appear to be somewhat high at pH 6.

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DISCUSSION

Primarily because of the ease with which light can be measured, bioluminescent assays are in general simple, rapid, and highly sensitive. Consequently, bioluminescence has been useful as an assay tool. For example, the firefly luciferase system is routinely used in the determination of low levels of ATP in crude extracts (7)) and aequorin has been used for the detection of CaZ+in viva (8). Bacterial luciferase provides a generally applicable, simple, rapid, and highly sensitive bioluminescent assay for protease activities. The effect of proteases on firefly luciferase activity was also investigated. It was inactivated 40 times more slowly than the bacterial enzyme. While this makes t,he firefly luciferase unsuitable for use in a protease assay, it implies that the firefly system ATP assay should be rather insensitive to proteolytic activity which might be present in crude tissue homogenates. The bacterial luciferase which was obtained commercially had a specific activity of only about 2<$ of our preparation. It was. however, rapidly inactivated by proteases in a fashion comparable to that observed with the pure material. Also, the luciferase in crude lysates of luminous bacteria is similarly suitable for the protease assay. One precaution that should be taken pertains to the concentration of luciferase. The rate of proteolytic inactivation of luciferase is dependent not only on the protease concentration, but also on the luciferase concentration, the rate being more rapid in less concentrated luciferase solutions, using pure material. In the concentration range of 50 /lg luciferase/ml, the inactivation rate is relatively independent of luciferase concentration. If the same concentration of luciferase is used for each assay, the inactivation rate will be proportional to the protease concentration. The effect of luciferase concentration appears to be indcpendent of the nature of the protease involved. Bacterial luciferase, which produces blue-green light (A,,, N 495 nm) by catalyzing the oxidation of FMNH, and a long-chain saturated aldehyde, is a dimeric protein (4) in which the active site appears to he on the Q subunit (9,lO). Over the time period needed to give 99% inactivation by trypsin, the (Ysubunit is converted from the native 42,0() dalton form (11) to a 28,000 dalton peptide plus smaller fragments, while the p subunit appears to be unaffected (1). Denatured proteins exist in many conformations and are therefore subject to proteolytic attack in a vast number of positions not accessible in the native conformation. Because proteolysis of denatured proteins involves cleavage at many different sites, it might be expected that prot,ease assays utilizing denatured substrates will be little affected by

286

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HASTINGS

specific substrate conformational changes induced by variations in pH or ionic strength. The luciferase method, on the other hand, employs a substrate in its native conformation. Presumably, the proteasesensitive region represents an exposed and flexible part of the peptide chain (12)) so the possibility of substrate effects cannot be ignored when comparing protease activities under different conditions. We have found, however, that the luciferase assay does not differ substantially from the casein and azocoll assays over the pH range 6-8. The requirement for luciferase activity precludes the use of agents in the protease assay which might directly inhibit or inactivate luciferase. Among these, mercurials (such as phenyl mercuric acetate) and other reagents which might be used as inhibitors of cysteine proteases are potent inhibitors of luciferase (13). Phenyl methyl sulfonyl fluoride, an inhibitor of serine proteases, does not affect luciferase. Acidic conditions will inactivate luciferase making this protease assay unsuitable for acid proteases. A wide variety of protease assays are currently available, each with particular advantages and shortcomings. Artificial substrates, especially synthetic amino acid esters, provide sensitive and convenient assays. Several spectrophotometric methods have been described, sensitive to as little as 200 ng trypsin (14-16). Roffman et al. (17) have described a method using a radioactive substrate which they state is capable of detecting as little as 1 ng of trypsin. These synthetic substrates are generally very convenient, requiring no centrifugation, but they are also quite specific and cannot be used to assay a wide variety of proteases. General protease assays using protein substrates customarily measure an increase in TCA soluble material. The method devised by Anson (4) has been made more sensitive through the use of radioactive or fluorescent substrates. 1311 labeled casein is capable of detecting 1 p.g/ml trypsin (18). Fluorescein-labeled hemoglobin is sensitive to about 209 ng trypsin (19). Solubilization of dyes attached to insoluble proteins provides a method capable of assaying 500 ng trypsin (3,20). These assays are all subject to the inconvenience of centrifugation needed to separate the proteolytic products from the substrate. The use of fluorescamine to label liberated amino groups avoids this problem (21,22), but can be complicated by high background due to free amino groups in the protease preparation. An assay of this type using protamine sulfate from herring as substrate is sensitive to 5 rig/ml trypsin (21). Protamine was selected for the trypsin assay because it contains 67% arginine. The method may not be that sensitive to proteases with other specificities. The luciferase assay is comparable to all of these assays in sensitivity and is among the most widely applicable. It is also convenient and does

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not require the centrifugation or the radioactivity safety precautions that many of the other assays require. It is not subject to interference from contaminating pigments as are the absorbance assays and to a lesser extent the fluorescence assays. It is vulnerable to agents or eontiitions which inhibit or inactivate luciferase or affect its native conformation. Because of its convenience, this assay should be especially useful in situations where many samples must be routinely assayed for proteolytic activity. Because of its generality, it should be helpful in the first stages of research on a proteolytic activity, before a specific artificial substrate can be found. Because of its immunity to background complications, it may be particularly useful in following the first steps in purification of proteases. REFERENCES 1. 2. 3. 4. 5.

BALDWIN, T. O., (1974) Fed. Proc. 33, 1441. HASTINGS, J. W., AND GIBSON, Q. H. (1963) J. Biol. Chem. 238, 2537. MOORE, G. 1,. (1969) Anal. Biochem 32, 122. ANSON, M. L. (1938) J. Gen. Physiol. 22, 79. G~~~~.~LLJ~-MI~~EL, A., MEIGHEN, E. A., NICOLI, M. Z., NEALSON, K. HASTINGS, J. W. (1972) J. Biol. Chem. 247, 398. 6. MITCHELL, G. W., AND HASTINGB, J. W. (1971) Anal. Biochem. 39, 243. 7. STREHLER, B. L., AND TOTTER, J. K. (1954) in Methods of Biochemical

Bnalysis D., ed.), Vol. I, p. 341, Interscience, New York. ASHLEY, C. C., AND RJDGWAY, E. B. (1968) Nature (London) 219, 1168. CLINE, T. W., AND I&STINGS, J. W. (1972) Biochemistry 11, 3359, MEIGHEN, E. A., NICOLI, M. Z., AND HASTINGS, J. W. (1971) Biochemistry 10, 4062, 4069. HASTINGS, J. W., WEBER, K., FRIEDLAND, J., EBERHARD, A., MITCHELL, G. UT., ASL) GUNSALUS, A. (1969) Biochemistry 8, 4681. NASLIN, L., SPYRIDARIS, A., AND LABEYRIE, F. (1973) Ew. J. Biochem. 34, 268. NICOLI, M. Z., MEIGHEN, E. A., AND HASTINGS, J. W. (1974) J. Biol. Chem. 249, 2385. SCHWERT, G. W., AND TAKENAKA, Y. (1955) Biochim. Biophys. A&I 16, 570. HUMMEL, B. C. W. (1959) Can. J. Biochem. Physiol. 37, 1393. ERLANGER, B. F., KOKOWSKY, N., AND COHEN, W. (1961) Arch. Biochem. Biophys. 95, 271. ROFFMAN, S., SANOCKA, U., AND TROLL, W. (1970) Anal. Biochem. 36, 11. KATCHMAN, B. J., ZIPF, R. E., AND HOMER, G. M. (1960) Nalltre (Lonr[on) 185, 238. DELUMEN, B. O., AND TAPPEL, A. 1~. (1970) Anal. Biochem. 36, 22. NELSON, W. L., CIACCIO, E I., AND HESS, G. P. (1961) Anal. B&hen&. 2, 39. BROWN, F., FREEDMAN, M. L., AND TROLL, W. (1973) Biochem. Biophys. Rea. Commun. 53, 75. SCHWARE, C. (1973) Anal. Biochem. 53, 484. (Click,

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22.

H., AND