Introduction

Human mast cells contain three serine proteases in their granules that are released upon degranulation. Most, if not all, mast cells contain tryptase, and some mast cells also contain chymase (1). Additionally, cathepsin G, which is normally thought of as a component of neutrophils, also has been shown to be present in mast cells (2,3). The activities of these proteolytic enzymes add additional functions to the mast cell. Consequently, the measurement of these enzyme activities is critical to studying mast cell function. Both chymase and CatG are chymotrypsin-like enzymes and distinguishing between their activities is difficult. Although tryptase is the only trypsin-like protease in mast cells, CatG also cleaves synthetic peptide substrates used to measure tryptase (4). Consequently, one needs to understand the subtle differences in the activities of these three enzymes and their interactions with inhibitors to measure their

From: Methods in Molecular Biology, vol. 315: Mast Cells: Methods and Protocols Edited by: G. Krishnaswamy and D. S. Chi © Humana Press Inc., Totowa, NJ

activities. For example, tryptase requires heparin for its activity. Although tryptase is very stable in solutions containing 2 M NaCl, it is rapidly inactivated in buffers of physiological ionic strength in the absence of heparin. Additionally, CatG is inhibited by heparin.

Tryptase P is a well-established marker of human mast cell activation or degranulation (5), and measurement of its enzymatic activity provides a simple method of detecting this process (6-8). Measurement of tryptase activity works well with purified mast cells, but one must be aware that other trypsin-like proteases may cleave the synthetic peptide substrate used to measure tryptase activity and interfere.

Protocols for the assay of each enzyme will be given along with information to aid in distinguishing the activity of each enzyme with a particular substrate. Although a number of para-nitroanilide and fluorescent peptide substrates have been used to assay mast cell proteases, thiobenzyl ester substrates are more sensitive than para-nitroanilide substrates and easier to use than fluorescent substrates. Castillo et al. (9) were the first to demonstrate the sensitivity and utility of peptide thiobenzyl ester substrates and they developed several very useful substrates for measuring protease activities. Proteases cleave thiobenzyl esters at much higher rates than para-nitroanilides, yet the thiobenzyl esters are far less sensitive to autolysis at alkaline pH than ethyl or methyl esters (10). Additionally, the benzene thiol product reacts with 5,5' dithiobis (2-nitro benzoic acid) (DTNB), also referred to as Ellman's reagent, to yield a yellow product (NBS [2-nitro-5-thiobenzoate anion]) that has a molar extinction coefficient of 13,600 cm-1 at 410 nm. The chemistry of these reactions is shown in Fig. 1. The sensitivities of thiobenzyl ester substrates relative to para-nitroanilide substrates can be seen by comparing kcat/Km values (substrate specificity constants) for the same peptide substrates (Table 1). For chymase the kcat/Km value with Succinyl-Ala-Ala-Pro-Phe-para-nitroanilide is 1 x 106 M-1s-1, whereas with Suc-AAPF-SBzl it is 16 x 106 M-1s-1, making the later substrate 16-fold more sensitive. Fluorescent substrates for tryptase have been developed with a 7-amino-4-carbamoylmethylcoumarin (ACC) fluorescent leaving group (11). The most specific of these substrates was N-acetyl-Pro-Arg-Asn-Lys-AAC (Ac-PRNK-ACC) with a reported kcat/Km of 1.23 x 106 M-1s-1, which is only slightly higher than 0.836 x 106 M-1s-1 found for the simple Z-Lys-SBzl (12). However, it must be pointed out that Ac-PRNK-ACC has the advantage that it is not readily cleaved by other trypsin-like enzymes, such as thrombin. By choosing the most specific peptide substrates and by using inhibitors to block interfering activities it is possible to assay different mast cell proteases. As knowledge increases regarding the specificities of these proteases more specific substrates will be developed.

Specific Substrate Assay Proteases Amp

Fig. 1. The chemistry of thiolbenzyl ester substrate assays.

Table 1

Thiolbenzyl Ester Substrate Kinetic Constants

Table 1

Thiolbenzyl Ester Substrate Kinetic Constants

Protease

Substrate

km (pm)

kcat (s 1)

kcat/km 10-6 X -Is-1

Ref

Tryptase

Z-K-SBzl

55

46

0.836

(12)

Chymase

Sus-AAPF-SBzl

22

363

16

(27)

Chymase

Suc-VPF-SBzl

48

400

8.3

(28)

CatG

Sus-AAPF-SBzl

48

14

0.3

(27)

CatG

Suc-VPF-SBzl

19

22

1.2

N-carbobenzoxy-Lysine thiobenzyl ester (Z-Lys-SBzl). Succinyl-Ala-Ala-Pro-Phe-thiobenzyl ester (Suc-AAPF-SBzl). DTNB. Heparin.

Visible Plate Reader with kinetic capability at 405 or 410 nm.

Tryptase Assay Buffer: 0.1 M HEPES, 10% glycerol v/v, 0.1 mg/mL Heparin,

0.01% Triton X-100 v/v, 0.02% NaN3 w/v, pH 7.5 (see Note 1).

20 mM Z-Lys-SBzl; dissolve 17 mg in 2 mL of isopropyl alcohol (warming the tube in hot water aids dissolution; see Note 2).

8. 10 mM solution of DTNB; dissolve 40 mg in 10 mL of Tryptase Assay Buffer. The DTNB solution is stable for approx 1 wk when stored at 4°C, but if it becomes visibly yellow it should be discarded.

9. Tryptase Substrate Assay Solution; mix 0.87 mL of Tryptase Assay Buffer, 0.1 mL of DTNB solution, and 30 pL of the 20 mM Z-Lys-SBzl stock solution, along with 1 pL of octanol to prevent bubble formation (see Note 3).

10. Chymase Assay Buffer: 0.1 M HEPES, 1 M NaCl, 10% glycerol v/v, 0.1 mg/mL heparin, 0.01% Triton X-100 v/v, 0.02% NaN3 w/v, pH 7.5. Because glycerol volumes are not easily measured, weigh 25 g of glycerol (density is 1.25 g/mL) into a 250-mL bottle on a top loading balance. Then add 150 mL of deionized water and mix before adding solids; 4.76 g of HEPES, 11.7 g of NaCl, 20 mg of heparin, 0.2 mL of 10% Triton X-100 in water, 40 mg of NaN3, adjust pH to 7.5 with NaOH and dilute to 200 mL (see Notes 1 and 4).

11. 20 mM stock solution of the Suc-AAPF-SBzl chymase substrate; dissolve 12.2 mg in 1 mL of dimethylsulfoxide (DMSO; not soluble in isopropanol) and store at -20°C (see Note 5).

12. 10 mM solution of DTNB; dissolve 40 mg in 10 mL of Chymase Assay Buffer. The DTNB solution is stable for approx 1 wk when stored at 4°C, but if it becomes visibly yellow it should be discarded.

13. Chymase Substrate Assay Solution; mix 0.87 mL of Assay Buffer, 0.1 mL of DTNB solution, and 30 pL of the 20 mM Suc-AAPF-SBzl stock solution, along with 1 pL of octanol to prevent bubble formation (see Note 3).

3. Methods

3.1. Assay of Tryptase Activity

1. Pipet samples containing tryptase into microplate wells and dilute to 50 pL with Tryptase Assay Buffer.

2. Pipet 50 pL of buffer in Blank wells in place of sample.

3. Set up the plate reader software to read the desired plates at 405 or 410 nm in kinetic mode.

4. Set the plate reader to mix on high speed for 10 s prior to the first read. Readings at 410 nm are normally taken at 20-s intervals for 10 min.

5. Start assay reactions by quickly adding 50 pL of Substrate Assay Solution.

6. Readings at 405 or 410 nm are normally taken at 20-s intervals for 10 min, but these parameters can be adjusted based on the amount of enzyme present (see Note 6).

7. A plot of initial rates versus the amount of enzyme should be linear as shown in Fig. 2.

3.2. Assay of Chymase Activity

1. Follow the steps in Subheading 3.1. for assaying tryptase using the Chymase Substrate Assay Solution containing Suc-AAPF-SBzl and the Chymase Assay Buffer in place of the solution used to assay tryptase.

Tryptase Standard Curve

0 200 400 600 800 1000 1200

Fig. 2. Tryptase standard curve. Recombinant human tryptase beta was assayed using the Z-Lys-SBzl substrate as described. Rates were plotted against the amount of enzyme.

0 200 400 600 800 1000 1200

Femtomoles of Tryptase

Fig. 2. Tryptase standard curve. Recombinant human tryptase beta was assayed using the Z-Lys-SBzl substrate as described. Rates were plotted against the amount of enzyme.

3.3. Application of the Assays to Human Mast Cells

1. The human leukemia mast cells lines HMC-1 (13) and a subclone of this cell line, designated 5C6 (14), were analyzed for their content of tryptase and chymase activities. Cord blood-derived mast cells (CBDMCs) also were analyzed for these enzyme activities. The cells were generously provided by Drs. D. Chi and G. Krishnaswamy of East Tennessee State University (15,16).

2. To measure the protease activities in cultured mast cells extracts of CBDMCs, HMC-1, and 5C6 cells were prepared. Preliminary work had shown that tryptase is very stable in high salt buffers at pH 6.1 and that Triton X-100, a nonionic detergent extracts additional tryptase and chymase activity from HMC-1 cells. Therefore, 10 mM MES; 2 M NaCl; 10% glycerol (v/v); 0.02% NaN3, pH 6.1; with 0.1% Triton X-100 was chosen as the cell extraction buffer. One tube of each of HMC-1, 5C6 (10 x 106 cells each), and CBDMC (1 x 106 cells), which had been stored as frozen cell pellets, was suspended in 2 mL of cold extraction buffer. Cell suspensions were transferred to 10 mL of polypropylene tubes and homogenized using an Omni 2000 rotary tissue grinder at maximum speed for 10 s and placed on ice. The rotary tissue grinder breaks the cells and reduces the viscosity of the extracts by sheering the DNA. Homogenates were stored overnight at 4°C and soluble fractions were obtained by centrifuging the samples in the same 10-mL tubes in the Beckman R-20 rotor using rubber adaptors at 30,000g for 30 min. Each extract represented 5 x 106 cells/mL for HMC-1 and 5C6 cells and 0.5 x 106 cells/mL for CBDMCs. Assays for tryptase and chymase

Fig. 3. Tryptase and chymase activities in mast cells. Extracts of two mast cell lines (HMC-1 and 5C6; 5 x 106 cells/mL) and CBDMC (0.5 x 106 cells/mL) were assayed for tryptase using Z-Lys-Bzl as described (A) and chymase using Suc-AAPF-SBzl (B) as described. Assays were performed in triplicate and the error bars are standard deviations from the mean.

Fig. 3. Tryptase and chymase activities in mast cells. Extracts of two mast cell lines (HMC-1 and 5C6; 5 x 106 cells/mL) and CBDMC (0.5 x 106 cells/mL) were assayed for tryptase using Z-Lys-Bzl as described (A) and chymase using Suc-AAPF-SBzl (B) as described. Assays were performed in triplicate and the error bars are standard deviations from the mean.

activities were performed on 10-40 pL of extract, using the Z-Lys-SBzl substrate for tryptase and the Suc-AAPF-SBzl substrate for chymase. The results of these assays are shown in Fig. 3A and 3B, respectively. Although CatG has been reported in human tissue-derived mast cells (2,3) and in the HMC-1 cell line (17), the amount of CatG relative to chymase is unknown. Because, on the basis of their kcat/Km ratios, Suc-AAPF-SBzl is a 53-fold better substrate for chymase than for CatG, it can be concluded that most of the activity observed is attributable to chymase even if these chymotrypsin-like enzymes are present in equal concentrations. Additionally, HMC-1 cells seem to have much less CatG than chymase (17), further indicating that the Suc-AAPF-SBzl substrate measures chymase, rather than CatG in this cell line. Although CatG activity might interfere with the measurement of tryptase and chymase activities, CatG does not cleave these synthetic substrates very rapidly and the amount of CatG in mast cells is probably low. Heparin is required for the stability of the active tryptase tetramer (12,18) and chymase is activated by heparin (19). Whereas heparin has been shown to inhibit CatG (20,21), the inclusion of heparin in the tryptase and chymase assay buffers would further reduce CatG activity.

3. Inhibitors can aid in distinguishing between different proteases, as demonstrated by Sheth et al. (17), who used aprotinin (bovine pancreatic trypsin inhibitor; also known as Trasylol a product of Bayer, AG), which inhibits CatG, but not chymase. These workers also used a synthetic inhibitor of chymase called Y-40018 (22) that inhibits chymase by 100% and CatG by only approx 20%. As pharmaceutical companies develop new inhibitors of chymase and/or CatG as potential drugs, more specific inhibitors will become available. Tryptase is uniquely resistant to inhibition by the protease inhibitors present in human blood (23) and this property can be used to distinguish tryptase from other trypsin-like serine proteases.

4. As with any enzyme assay, running positive controls with known amounts of pure enzyme aids in standardizing the technique and for testing its application to the system of interest. Fortunately, recombinant human tryptase (24) is available from Promega (Madison, WI). Chymase and CatG purified from human tissues are commercially available, although recombinant forms not yet available. Active recombinant chymase has been expressed (25), but there are no reports of recombinant CatG.

4. Notes

1. Because glycerol volumes are not easily measured, weigh 25 g of glycerol (density is 1.25 g/mL) into a 250-mL bottle on a top of a loading balance. Then, add 150 mL of deionized water and mix, before adding solids, 4.76 g of HEPES, 20 mg of heparin, 0.2 mL of 10% Triton X-100 in water, 40 mg of NaN3, adjust pH to 7.5 with NaOH, and dilute to 200 mL. As mentioned earlier, heparin is required for tryptase stability, and we have found that glycerol also aids enzyme stability. Triton X-100 prevents the tendency of tryptase to bind to surfaces at lower ionic strengths.

2. Although Z-Lys-SBzl stock solutions can be prepared in DMSO, we have found that the substrate gives lower rates over time when stored in DMSO, whereas this does not happen when the isopropanol is used as the solvent. There was no indication that the substrate was hydrolyzing during storage in DMSO because the substrate buffer blanks did not increase during storage. Although we have not analyzed the chemistry associated with this loss of substrate function in DMSO, we do know that Z-Lys-SBzl is very stable in isopropaol.

3. The volume of Substrate Assay Solution prepared depends on the number of wells being used plus enough extra volume to allow pipetting. For example to assay 20

wells make up 1.2 mL of Substrate Assay Solution. A multichannel pipetor is useful for quickly adding the Substrate Solution when assaying a large number of wells, but there is more substrate wastage because extra volume must be in the buffer troughs used with multichannel pipetors.

4. The assay buffer for chymase is essentially the same as that used for tryptase with the addition of 1 M NaCl, which has been shown to increase activity (26).

5. Suc-AAPF-SBzl has a 53-fold larger kcat/Km ratio for chymase than for CatG and the addition of heparin to the buffer activates chymase and inhibits CatG. Consequently, assays of mast cell extracts using the Suc-AAPF-SBzl substrate predominantly measure chymase with little interference from CatG.

6. Although we normally perform assays at room temperature (approx 22°C), higher temperatures, such as 37°C can be used. To obtain initial velocity measurements check the linearity of each reaction via linear regression. When the curves are not linear over the course of 10 min, initial velocities (or rates) usually can be obtained by using the data from the first 2 to 5 min. If the plots are not linear, repeat the assays using less enzyme (cell extract) so the initial velocities can be accurately determined.

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