DSpace at VNU: Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane - Tricine buffer system

Detection of protease inhibitors by a reverse zymography method, performed in a trishydroxymethylaminomethane–Tricine buffer system a Institute for Health Sciences, Tokushima Bunri Univer

Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane–Tricine

buffer system

a Institute for Health Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima City, Tokushima 770-8514, Japan

b Biotechnology Center, Vietnam National University, Hanoi 144 Xuan thuy-Cau giay, Hanoi, Vietnam

Received 22 July 2003

Abstract

A new detecting method for protease inhibitors, especially for low-molecular-weight inhibitors, is reported Inhibitor samples were separated on a protein substrate–SDS–polyacrylamide gel in a Tris–Tricine buffer system that improves the separation and identification of peptides and low-molecular-weight proteins After electrophoresis, the gel was incubated with the target proteases

to hydrolyze the background protein substrate The inhibitor bands, which were protected from proteolysis by the target proteases, were stained Standard low-molecular-weight inhibitors, such as pepstatin A for pepsin or matrix metalloproteases inhibitor I for collagenase, as well as larger inhibitors, such as soybean trypsin inhibitor or aprotinin for tryspin and cystatin C for papain, were demonstrated by this method and showed clear blue inhibitor bands in the white background when the gels were treated with the target proteases Some significant applications of this method are introduced This method is an ideal system for discovering new protease inhibitors in small natural samples

Ó 2003 Elsevier Inc All rights reserved

Keywords: Reverse zymography; Protease inhibitors

Endogenous protease inhibitors are potential key

regulators of proteases in living organisms Reverse

zy-mography techniques are effective tools for isolating and

characterizing natural protease inhibitors, particularly in

medical science [1–4] These methods are based on the

separation of the inhibitor samples on an

SDS–poly-acrylamide gel containing protein substrate such as

gel-atin or casein copolymerized into the gel After

electrophoresis, washing and incubating the gel with the

target protease solution at 37°C for an optimal

incuba-tion period allow the substrate to be digested by the

target proteases The undigested protein substrate

re-mains where the inhibitor molecules are located and can

be stained as blue bands These methods, although useful

in searching for numerous protease inhibitors, failed to

identify low-molecular-weight inhibitors We developed

a new reverse zymography method, performed in a Tris– Tricine buffer system [5], which allows better resolution

of peptides and low-molecular-weight proteins than the Tris–glycine system [5,6] The new method offers several significant advantages (i) it is quite simple and the in-hibitor bands can be visualized clearly in the gel, (ii) a wide range of different-molecular-weight inhibitors within protein pools could be selectively detected and (iii)

it allows the simultaneous determination of protease inhibitory activity and molecular weight and could be useful for discovering new protease inhibitors in small amounts of crude material We have successfully dis-covered various inhibitors in physiological materials using this reverse zymography technique

Materials and methods Reagents required for gel preparation, gelatin, stan-dard inhibitors such as cystatin C, pepstatin A, soybean

*

Corresponding author Fax: +81-88-622-2503.

E-mail address: katunuma@tokushima.bunri-u.ac.jp (N

Katu-numa).

0003-2697/$ - see front matter Ó 2003 Elsevier Inc All rights reserved.

doi:10.1016/j.ab.2003.09.033

Analytical Biochemistry 324 (2004) 237–240

ANALYTICAL BIOCHEMISTRY

www.elsevier.com/locate/yabio

trypsin inhibitor (STI),1 aprotinin, matrix

metallo-proteases inhibitor I (MMPI), and other materials, all

pure grade, were purchased from Sigma Chemical

Co.(St, Louis, MO, USA) Molecular weight markers

were obtained from Bio-Rad Chemical Co (Richmond,

CA, USA) All other reagents used were of analytical

grade

Solutions for making gels were prepared according to

Schagger and Von Jagow [5] with some modification

The separating gel, consisting of 0.1% (w/v) gelatin, 15%

(w/v) acrylamide, 0.4% (w/v) bis-acrylamide, 10% (v/v)

glycerol, 0.75 M Tris–HCl (pH 8.45), and 0.1% SDS,

was cast in a Hoefer gel cassette (Pharmacia Biosciences

Co Upsala, Sweden) with gel dimensions of

8 10  0.1 cm In some experiments, to compare with

the gelatin gel, 0.1% collagen or hemoglobin was used in

the gels Then a stacking gel of a mixture of stock

so-lution of 4% (w/v) acrylamide, 0.14% (w/v)

bisacryla-mide, and 330 mM Tris–HCl, pH 6.8 (no substrate), was

cast with sample combs on top of the separating gel

The authentic inhibitors or human tears were mixed

with an equal volume of a treatment buffer (nonreducing

reagent) of 4.0% SDS, 20% (v/v) glycerol, 0.25 M Tris–

HCl, pH 6.8, and 0.02% bromophenol without heat

treatment Samples of 15 ll were applied to the sample

wells

The electrophoresis was performed in a Tris–Tricine

buffer system [0.2 M Tris (pH 8.9) as an anode buffer

and 0.1 M Tris, 0.1 M Tricine, 0.1% SDS (pH 8.25) as a

cathode buffer] as described by Schagger and Von Jagow

[5] at 4°C at a constant current of 10–12 mA After the

electrophoresis was completed, the gel was removed and

shaken at room temperature for 45 min in 2.5% Triton

X-100 to remove SDS The gel was washed with distilled

water several times and incubated at 37°C in reverse

zymography developing buffer containing a target

en-zyme for 9–10 h to digest out the background substrate

The development buffer for soybean trypsin inhibitor or

aprotinin consisted of 10 mM Tris–HCl buffer (pH 7.6),

200 mM NaCl, 10 mM CaCl2, 0,02% Brij-35, and 450 lg

/100 ml trypsin

Cystatin C or human tears were developed in 10 mM

sodium acetate buffer (pH 6.1) containing 1 mg papain

(31 unit/mg of papain), MMPI was in 20 mM

phos-phate-buffered saline, pH 7.2, containing 1 mg /100 ml

collagenase type 1, and pepstatin A was in 20 mM

uni-versal Britton buffer, pH 3.0, containing 1 mg/100 ml

pepsin A After proteolysis by incubation in the protease

solutions, the gel was stained with staining solution

(0.025% Coomassie brilliant blue R250; 40% methanol,

7% acetic acid) for 1 h The gels were washed three times

for 1 h with destaining solution (40% methanol, 10% acetic acid, 50% distilled water.)

Result and discussion

To select the most suitable substrate for our reverse zymography technique, we compared the staining in-tensities of gelatin, collagen, and hemoglobin gels before and after electrophoresis without inhibitor samples As seen in Fig 1, the collagen, gelatin and hemoglobin gels were stained weak violet, purple-blue, and dark blue (lanes 1a and 1b, 2a and 2b, and 3a and 3b), respec-tively Both before and after electrophoresis, the stained collagen gel lost its color very quickly after 2 or 3 h

Fig 1 Comparison of the CBB binding of collagen, hemoglobin, and gelatin gels before and after electrophoresis without inhibitor samples Dye-stained gels were destained 12 h Lane 1, collagen gel: (a) before running, (b) after running Lane 2, gelatin gel: (a) before running, (b) after running Lane 3, hemoglobin gel: (a) before running, (b) after running.

Fig 2 Gelatin background intensity pattern and densitogram of soybean trypsin inhibitor after different incubation times (A) Gelatin background intensity during incubation period (B) Densitogram of soybean trypsin inhibitor after a 10-h incubation.

1 Abbreviations used: SDS, sodium dodecyl sulfate; PAGE,

poly-acrylamide gel electrophoresis; CBB, Coomassie brilliant blue; STI,

soybean trypsin inhibitor; MMPI, matrix metalloproteases inhibitor I.

238 Q.T Le, N Katunuma / Analytical Biochemistry 324 (2004) 237–240

(lanes 1a and 1b), while the gelatin and hemoglobin gels

maintained their color until 12 h (lanes 2a and 2b and 3a

and 3b) Furthermore, the stained color was not

ho-mogeneous in the case of the hemoglobin gel, while the

gelatin gel could retain its purple-blue color

homoge-neously throughout the gel (lane 2b) after

electropho-resis We observed the strongest Coomassie blue

staining intensity toward the end of the hemoglobin gel

run, which might be due to the hemoglobin migrating

out of the gel during electrophoresis (lane 3b) Based

upon our observation above that gelatin was sensitive to

dye staining and did not migrate out from the gel, we

chose gelatin as the best substrate for our reverse

zy-mography technique

To extend the scope of this new reverse zymography

technique, different incubation times (from 30 min to

15 h) were evaluated using STI as a sample Fig 2 shows

that the optimal reverse zymography pattern giving the

sharpest STI band on the background gel was obtained

after 8–10 h After longer incubation times, the STI

band gradually disappeared

To determine which types of inhibitors can be iden-tified by our technique, we analyzed pepstatin A, MMPI, STI, aprotinin, and cystatin C on a 15% SDS– polyacrylamide gel containing 0.1% gelatin made by following the well- known Hanspal et al method [1] and our method running in Tris–glycine and Tris–Tricine system buffers, respectively After running, the gels from both procedures were treated under the same conditions

as described under Materials and methods Surprisingly, the inhibitor activity of 10–20 ng of cystatin C, STI, or aprotinin was detected as a sharp single inhibitor band

on the stained gels by both methods with an apparent molecular mass of 15, 25, or 12 kDa (Figs 3A and B, lanes 1–3), respectively Thus, these inhibitors showed similar molecular sizes using both methods; however, their migration was slightly slower by our method This result suggested that our method is suitable for detecting common protease inhibitors

The previously reported method [1] based on the Laemmli buffer system [7] has been preferred for the study of common protease inhibitors However, as can

be seen in Fig 3A, it afforded poor resolution for de-tecting small inhibitors under 1 kDa such as pepstatin A (685 Da) or MMPI (490 Da) They could not be detected

in the gel (lanes 4 and 5, Fig 3A) In contrast, in our reverse zymography method running in the Tris–Tricine buffer system, the resolution was considerably improved compared with Hanspal et al.Õs method In the new method, 5 10 7M pepstatin A and MMPI were visu-alized as clear inhibitor bands on the gel against pepsin

A or collagenase type 1, respectively (lanes 4 and 5, Fig 3B), indicating the advantage of this technique for detecting low-molecular-weight inhibitors These results

Fig 3 Comparison of the reverse zymography pattern of standard

inhibitors according to Hanspal et al.Õs method with those of our

method Slab gels consisted of 15% acylamide, 0.1% SDS, and 0.1%

gelatin Samples were subjected to electrophoresis: lane 1, cystatin C

(10 ng); lane 2, soybean trypsin inhibitor (15 ng); lane 3, aprotinin

(10 ng); lane 4, MMPI type 1 (5  10 7 M); lane 5, pepstatin A

(5  10 7 M) (A) Electrophoresis was performed in the Tris–glycine

buffer (B) Electrophoresis was performed in the Tris–Tricine buffer.

After running, the gels from both procedures were developed under the

same conditions as described under Materials and methods.

Fig 4 Inhibitors in normal human tears using reverse zymography to papain Lane 1, Coomassie blue staining of all proteins in human tear Lane 2, new reverse zymography pattern of papain inhibitors in human tear.

Q.T Le, N Katunuma / Analytical Biochemistry 324 (2004) 237–240 239

were in agreement with those of Shagger and Von Jagow

[5], who pointed out that separation of peptides and

small proteins are not possible in the Tris–glycine buffer

because the comigration of SDS and smaller protein

obscures the resolution Using the Tris–Tricine buffer

system in which Tricine was substituted for glycine as

the trailing ion in the running buffer of the new reverse

zymography method reported here allows the separation

of small inhibitors–SDS complexes away from the

rap-idly moving SDS micelles, providing distinct small

in-hibitor bands in the gel

Fig 4 shows the reverse zymography pattern for

cysteine protease inhibitors in human tears There were

three major inhibitor bands with molecular sizes of 78,

18, and 15 kDa, as shown in lane 2 The

high-molec-ular-weight inhibitor of 78 kDa was identified as a

lactoferrin using intramolecular amino acid sequence

analysis [8] The peptide sequences of 18 amino acids in

the near C-terminals area of lactoferrin showed 89%

homology and 61% identity with amino acid sequence

of a common active site of the cystatin family [8] The

finding of lactoferrin as a new cysteine protease

in-hibitor has not been reported in the literature

Addi-tionally, the 15 and 18-kDa of low-molecular-weight

inhibitor bands were clearly separated and these

in-hibitors were identified as well-known cysteine

prote-ases: a cystatin S [9] and von EbnerÕs gland protein

[10], respectively

In summary, this reverse zymography method is

useful for identifying both low- and

high-molecular-weight inhibitors against a variety of proteolytic

enzymes in small amounts of natural materials

Fur-thermore, we can compare the changes in these inhibitor

profiles in various biological and pathological

condi-tions using this reverse zymography

Acknowledgment

We thank Dr H Tsuge for helpful discussions

References

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[3] D.S Seidl, W.G Jaffe, E Gonzalez, A Callejas, Microelectro-phoretic method for the detection of proteinase inhibitors, Anal Biochem 88 (2) (1978) 417–424.

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[10] P Wojnar, W Vant Hof, P Merschak, B Redl, The N-terminal part of recombinant human tear lipocalin/von EbnerÕs gland protein confers cysteine proteinase inhibition depending on the presence of the entire cystatin-like sequence motifs, Biol Chem.

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