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Enzyme Manual: Elastase

I.U.B.: 3.4.21.36

C.A.S.: 9004-06-2 

Enzymatic Reaction (image will open in a new window) 

Elastase is a serine protease that also hydrolyzes amides and esters. It is produced in the pancreas as an inactive zymogen, and activated in the duodenum by trypsin. The following information applies to porcine elastase.

History:

Eijkman is believed to be the first to have studied elastase as a product of bacteria (Eijkman 1904). However, it was not shown to differ from other proteolytic components of the pancreas, such as trypsin and chymotrypsin, until the Baló and Banga publication in 1949.

In 1968, Shotton and Hartley were the first to obtain crystalline porcine elastase. In the late 1960s and into the early 1970s, elastase’s involvement in emphysema, atherosclerosis, and acute hemorrhagic pancreatitis was investigated (Janoff and Sherer 1968, Janoff and Basch 1971, Talamo et al. 1971, Kaplan et al. 1973, and Bieth et al. 1974). In 1974, Ardelt discovered a second elastolytic proteinase, naming it pancreatic “elastase II” and the original “elastase I”.

In the 1980s, the catalytic properties of elastase were studied (Ascenzi et al. 1983), and inhibitors were identified (Largman et al. 1980, Lestienne et al. 1981, Kettner and Shenvi 1984, Poncz et al. 1984, and Imperiali and Abeles 1986). Elastase I and II genetic information was studied into the 1990s (Ornitz et al. 1985, Stevenson et al. 1986, Swift et al. 1984, Tani et al. 1987, and Gestin et al. 1997).

Elastase has recently been used in investigations of the mechanical forces and enzyme activity in extracellular matrix breakdown (Jesudason et al. 2010). Its use in artificial organ development has also been studied (Tedder et al. 2010), and it is used as a model to investigate the catalytic activity of the serine proteases (Tamada et al. 2009).

Specificity:

Porcine elastase I is specific for Ala-Ala and Ala-Gly bonds, while elastase II has a broad specificity for substrates with medium to large hydrophobic amino acids in the P1 position (Gertler et al. 1977, Del Mar et al. 1980, and Gestin et al. 1997). Porcine elastase is the most potent elastase, having a rate 20-fold higher than that of human leukocyte elastase (Bieth 1978, Bieth 1986, and Largman 1983).  

Hydrolysis occurs in several steps. An adsorption complex between elastase and its substrate is formed, followed by nucleophilic attack (S214) to form an acyl-enzyme intermediate, and release of the first product (the C-terminal end of the substrate). The intermediate is hydrolyzed in a deacylation step, regenerating the active enzyme and releasing the second product (Bieth 1986).

Composition:

The catalytic triad is formed by three hydrogen-bonded amino acid residues (H71, D119, and S214). The polypeptide chain is composed of two antiparallel beta-barrel domains, which form a crevice containing the catalytic triad, and a small proportion of alpha-helices (Bieth 2004). 

Molecular Characteristics:

Porcine pancreatic elastase is composed of a single peptide chain of 240 amino acids, and contains 4 disulfide bridges (Sawyer et al. 1973). It has a high degree of sequence identity with pancreatic elastases from other species, such as rat with whom it shares 86% identity (MacDonald et al. 1982). Elastase I and II genes share sequence similarity, especially in the 5’ proximal flanking regions, which include the TATA box and a putative tissue-specific enhancer sequence (Ornitz et al. 1985, Stevenson et al. 1986, Swift et al. 1984, Tani et al. 1987, and Gestin et al. 1997). 

Protein Accession Number: P00772

Molecular Weight:

  • 26.0 kDa (Bieth 2004)

Optimal pH:

  • 8.5

Isoelectric Point:

  • 9.5 (Bieth 2004)

Extinction Coefficient:

  • 54,870 cm-1 M-1 (Theoretical)
  • E1%, 280 = 21.18 (Theoretical)

Active Site Residues:

  • Histidine (H71)
  • Aspartic acid (D119)
  • Serine (S214)

Inhibitors:

  • Competitively inhibited by derivatives of dipeptides of alanine, valine, leucine, and isoleucine
  • Inhibitors from natural sources (e.g. serpins) (Tsunemi et al. 1996, Matern et al. 2003, and Dementiev et al. 2006) 
  • Modified natural inhibitors (Ay et al. 2003, and Hilpert et al. 2003)
  • Sulfate-modified lipids (Ito et al. 1998)

Applications:

  • Tissue dissociation: Because elastin is found in highest concentrations in the elastic fibers of connective tissues, elastase is frequently used to dissociate tissues that contain extensive intercellular fiber networks. For this purpose, it is usually used with other enzymes such as collagenase, trypsin, and chymotrypsin.
  • Membrane protein solubilization
  • Protein sequence studies 

Assay

Bieth et al. (1974) have described an excellent assay using succinyl-(L-alanine)3-p-nitroanilide. Other suggested assays have been described: Keller and Mandl (1971), Feinstein et al. (1973), and Bieth and Meyer (1973). Schumacher and Schill (1972) describe a radial diffusion assay. The assay used in this laboratory is as follows:

Method: Derived from that of Feinstein et al. (1973) and using the more soluble substrate of Bieth et al. (1974). An increase in absorbance at 410 nm results from the hydrolysis of N-succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (Suc Ala3NA). One unit hydrolyzes one micromole of Suc Ala3NA per minute at 25°C and pH 8.0 under the specified conditions.

Reagents

  • 0.1 M Tris buffer, pH 8.0
  • 0.0044 M Suc Ala3NA substrate dissolved in Tris buffer. (2 mg/ml)

Enzyme

Prepare a one mg/ml solution in Tris buffer. Immediately before use dilute further to obtain a rate of 0.02-0.04 ΔA/minute.

equation

Procedure

Adjust the spectrophotometer to 410 nm and 25°C.

Pipette into each cuvette as follows:

Tris buffer 2.7 ml
Enzyme 0.1 ml

Mix and incubate in the spectrophotometer 4-5 minutes to achieve temperature equilibration. To test cuvette add 0.2 ml of substrate, mix, and record increase A410 for 3-5 minutes. Calculate ΔA/minute from the linear portion of the curve.

Calculation

calculation

References

• Ascenzi, P., Menegatti, E., Guarneri, M., and Antonini, E.: Catalytic Properties of Porcine Pancreatic Elastase: A Steady-state and Pre-steady-state Study , Mol Cell Biochem 56, 33, 1983
 
• Atlas, D., and Berger, A.: Size and Stereospecificity of the Active Site of Porcine Elastase , Biochemistry 12, 2573, 1973
 
• Atlas, D., Levit, S., Schechter, I., and Berger, A.: On the Active Site of Elastase: Partial Mapping by Means of Specific Peptide Substrates , F.E.B.S. Lett. 11, 281, 1970
 
• Avrameas, S., and Uriel, J.: Systematic Fractionation of Swine Pancreatic Hydrolases. II. Fractionation of Enzymes Insoluble in Ammonium Sulfate Solution at 0.40 Saturation , Biochemistry 4, 1750, 1965
 
• Bagdy, D.: Effect of Elastase on Blood Coagulation. German , Experientia 15, 122, 1959
 
• Banga, I., and Ardelt, W.: Studies on the Elastolytic Activity of Serum , Biochim Biophys Acta 146, 284, 1967
 
• Baumstark, J., Bardawil, W., Sbarra, A., and Hayes, N.: Purification of Pancreatopeptidase E by Batch Separation on DEAE-Cellulose , Biochim Biophys Acta 77, 676, 1964
 
• Bender, M., and Marshall, T.: The Elastase-Catalyzed Hydrolysis of p-Nitrophenyl Trimethylacetate , J Am Chem Soc 90, 201, 1968
 
• Bieth, J., and Wermuth, C.: The Action of Elastase on p-Nitroanilide Substrates , Biochem Biophys Res Commun 53, 383, 1973
 
• Bieth, J., Collin-Lapinet, G., Robert, L.: Elastases: Structure, Function and Pathological Role, , S. Karger, , 1978
 
• Bieth, J., Spiess, B., and Wermuth, C.: The Synthesis and Analytical Use of a Highly Sensitive and Convenient Substrate of Elastase , Biochem Med 11, 350, 1974
 
• Boudier, C., Holle, C., and Bieth, J.: Stimulation of the Elastolytic Activity of Leukocyte Elastase by Leukocyte Cathepsin G , J Biol Chem 256, 10256, 1981
 
• Brown, J., Kauffman, D., and Hartley, B.: The Primary Structure of Porcine Pancreatic Elastase. The N-Terminus and Disulphide Bridges , Biochem J 103, 497, 1967
 
• Brown, W., and Wold, F.: Alkyl Isocyanates as Active-Site-Specific Inhibitors of Chymotrypsin and Elastase , Science 174, 608, 1971
 
• Chao, S., Sciarra, J., and Vosburgh, G.: A Spectrophotometric Method for Determination of Elastase and Serum Elastase Inhibitor , Proc Soc Exp Biol Med 109, 342, 1962
 
• Dvonch, W., and Alburn, H.: Zone Electrophoresis of Elastase Preparations on Starch , Arch Biochem Biophys 79, 146, 1959
 
• Dzialoszynski, L., and Hofmann, T.: Competitive Inhibition of Pancreatic Elastase , Biochim Biophys Acta 302, 406, 1973
 
• Feinstein, G., Kupfer, A., and Sokolovsky, M.: N-Acetyl-(L-Ala)3-p-Nitroanilide as a New Chromogenic Substrate for Elastase , Biochem Biophys Res Commun 50, 1020, 1973
 
• Frosco, M., Chase, T., and Macmillan, J.: Purification and Properties of the Elastase from Aspergillus fumigatus , Infect Immun 60, 728, 1992
 
• Fujimoto, K., Ogawa, M., Saito, N., Kosaki, G., Minamiura, N., and Yamamoto, T.: A Novel Method of Isolation and Some Characteristic Properties of Human Pancreatic Elastases , Biochim Biophys Acta 612, 262, 1980
 
• Gauthier, F., Fryksmark, U., Ohlsson, K., and Bieth, J.: Kinetics of the Inhibition of Leukocyte Elastase by the Bronchial Inhibitor , Biochim Biophys Acta 700, 178, 1982
 
• Geneste, P., and Bender, M.: Esterolytic Activity of Elastase , Proc Natl Acad Sci U S A 64, 683, 1969
 
• Geokas, M., Wilding, P., Rinderknecht, H., and Haverback, B.: Determination of Elastase in Mammalian Plasma and Serum , J Clin Invest 46, 1059, 1967
 
• Gertler, A., and Birk, Y: Isolation and Characterization of Porcine Proelastase , Eur J Biochem 12, 170, 1970
 
• Gertler, A., and Hofmann, T.: Acetyl-L-Alanyl-L-Alanyl-L-Alanine Methyl Ester: A New Highly Specific Elastase Substrate , Can J Biochem 48, 384, 1970
 
• Gold, R., and Shalitin, Y.: On the Specificity of Porcine Elastase , Biochim Biophys Acta 410, 421, 1975
 
• Gorbunoff, M., and Timasheff, S.: The Role of Tyrosines in Elastase , Arch Biochem Biophys 152, 413, 1972
 
• Grant, N., and Robins, K.: Studies on Porcine Elastase and Proelastase , Arch Biochem Biophys 66, 396, 1957
 
• Hall, D.: The Identification and Estimation of Elastase in Serum and Plasma , Biochem J 101, 29, 1966
 
• Hall, D., and Wilkenson, J.: Elastase and Thrombogenesis , Nature 197, 454, 1963
 
• Imperiali, B., and Abeles, R.: Inhibition of Serine Proteases of Peptidyl Fluoromethyl Ketones , Biochemistry 25, 3760, 1986
 
• Janoff, A., and Basch, R.: Further Studies on Elastase-Like Esterases in Human Leukocyte Granules , Proc Soc Exp Biol Med 136, 1045, 1971
 
• Janoff, A., Rosenberg, R., and Galdston, M.: Elastase-Like Esteroprotease Activity in Human and Rabbit Alveolar Macrophage Granules , Proc Soc Exp Biol Med 136, 1054, 1971
 
• Jordan, R., Hewitt, N., Lewis, W., Kagan, H., and Franzblau, C.: Regulation of Elastase-Catalyzed Hydrolysis of Insoluble Elastin by Synthetic and Naturally Occurring Hydrophobic Ligands , Biochemistry 13, 3497, 1974
 
• Kagan, H., Crombie, G., Jordan, R., Lewis, W., and Franzblau, C.: Proteolysis of Elastin-Ligand Complexes. Stimulation of Elastase Digestion of Insoluble Elastin by Sodium Dodecyl Sulfate , Biochemistry 11, 3412, 1972
 
• Kaplan, H., Symonds, V., Dugas, H., and Whitaker, D.: A Comparison of Properties of the alpha-lytic Protease of Sorangium sp. and Porcine Elastase , Can J Biochem 48, 649, 1970
 
• Kaplan, P., Kuhn, C., and Pierce, J.: The Induction of Emphysema with Elastase. I. The Evolution of the Lesion and the Influence of Serum , J Lab Clin Med 82, 349, 1973
 
• Katagiri, K., Takeuchi, T., Taniguchi, K., and Sasaki, M.: One Step Purification Procedure of Elastase from Pancreatic Powder by Affinity Chromatography , Anal Biochem 86, 159, 1978
 
• Keller, S., and Mandl, I.: Solubilized Elastin as a Substrate for Elastase and Elastase Inhibitor Determinations , Biochem Med 5, 342, 1971
 
• Keller, S., Levi, M., and Mandl, I.: Antigenicity and Chemical Composition of an Enzymatic Digest of Elastin , Arch Biochem Biophys 132, 565, 1969
 
• Largman, C., Brodrick, J., and Geokas, M.: Human Pancreatic Proelastase 2. Sequence of the Activation Peptide , Biochim Biophys Acta 623, 208, 1980
 
• Largman, C., Delmar, E., Brodrick, J., Fassett, M., and Geokas, M.: Inhibition of Human Pancreatic Elastase 2 by Peptide Chloromethyl Ketones , Biochim Biophys Acta 614, 113, 1980
 
• Ledoux, M., and Lamy, F.: Electrophoretic Characterization of Porcine Pancreatic (Pro)elastases A and B , Can J Biochem 53, 421, 1975
 
• Legrand, Y., Pignaud, G., Caen, J., Robert, B., and Robert, L.: Separation of Human Blood Platelet Elastase , Biochem Biophys Res Commun 63, 224, 1975
 
• Lestienne, P., Dimicoli, J., Wermuth, C., and Bieth, J.: A New Class of Synthetic Elastase Inhibitor , Biochim Biophys Acta 658, 413, 1981
 
• Lewis, U., and Thiele, E.: The Isolation of a Pancreatic Insulinase , J Am Chem Soc 79, 7555, 1957
 
• Lewis, U., Williams, D., and Brink, N.: Pancreatic Elastase: Purification, Properties, and Function , J Biol Chem 222, 705, 1956
 
• Lewis, U., Williams, D., and Brink, N.: Pancreatic Elastase: Differentiation from other Pancreatic Proteinases , J Biol Chem 234, 2304, 1959
 
• Magaro, M., Greco, A., Sensi, S., Gambassi, G., and Maggi, V.: Experimental Aspects of the Clinical Use of Elastase , JAMA 176, 191, 1961
 
• Mandl, I.: Pancreatic Elastase , Methods in Enzymology Vol. 5, S. Colowick and N. Kaplan, Academic Press, NY, 665, 1962
 
• Mason, R., Johnson, D., Barrett, A., and Chapman, H.: Elastinolytic Activity of Human Cathepsin L , Biochem J 233, 925, 1986
 
• Murata, A., Ogawa, M., Fujimoto, K., Kitahara, T., Matsuda, Y., and Kosaki, G.: Radioimmunoassay of Human Pancreatic Elastase 1. In vitro Interaction of Human Pancreatic Elastase 1 with Serum Protease Inhibitors , Enzyme 30, 29, 1983
 
• Narayanan, A., and Anwar, R.: The Specificity of Purified Porcine Pancreatic Elastase , Biochem J 114, 11, 1969
 
• Naughton, M., and Sanger, F.: Purification and Specificity of Pancreatic Elastase , Biochem J 78, 156, 1961
 
• Naughton, M.,Sanger, F., Hartley, B., and Shaw, D.: The Amino Acid Sequence around the Reactive Serine Residue of Some Proteolytic Enzymes , Biochem J 77, 149, 1960
 
• Powers, J., and Tuhy, P.: Active-Site Specific Inhibitors of Elastase , J Am Chem Soc 94, 6544, 1972
 
• Powers, J., and Tuhy, P.: Active-Site Specific Inhibitors of Elastase , Biochemistry 12, 4767, 1973
 
• Powers, J., Boone, D., Carroll, D., Gupton, B., Kam, C., Nishino, N., Sakamoto, M., and Tuhy, P.: Reaction of Azapeptides with Human Leukocyte Elastase and Porcine Pancreatic Elastase. New Inhibitors and Active Site Titrants , J Biol Chem 259, 4288, 1984
 
• Quinn, R., and Blout, E.: Spectrofluorometric Assay for Elastolytic Enzymes , Biochem Biophys Res Commun 40, 328, 1970
 
• Renaud, A., Lestienne, P., Hughes, D., Bieth, J., and Dimicoli, J.: Mapping of the S' Subsites of Porcine Pancreatic and Human Leucocyte Elastases , J Biol Chem 258, 8312, 1983
 
• Rinderknecht, H., Geokas, M., Silverman, P., and Haverback, B.: Determination of Elastolytic Activity in Blood of Normal Subjects and Patients with Acute Pancreatitis , Clin Chim Acta 19, 89, 1968
 
• Rinderknecht, H., Geokas, M., Silverman, P., Lillard, Y., and Haverback, B.: New Methods for the Determination of Elastase , Clin Chim Acta 19, 327, 1968
 
• Rinderknecht, H., Geokas, M., Swanson, V., and Haverback, B.: Pancreatic Elastase and Hemorrhagic Pancreatitis , Gastroenterology 56, 803, 1969
 
• Sachar, L., Winter, K., Sicher, N., and Frankel, S.: Photometric Method for Estimation of Elastase Activity , Proc Soc Exp Biol Med 90, 323, 1955
 
• Sawyer, L., Shotton, D., and Watson, H.: Atomic Coordinates for Tosyl-Elastase , Biochem Biophys Res Commun 53, 944, 1973
 
• Schellenberger, V., Schellenberger, U., Mitin, Y., and Jakubke, H.: Characterization of the S'-Subsite Specificity of Porcine Pancreatic Elastase , Eur J Biochem 179, 161, 1989
 
• Schneider, I., Tindel, S., Shapiro, D., and State, D.: Colorimetric Method of Measuring Pancreatic Elastase Activity , J Lab Clin Med 60, 514, 1962
 
• Shotton, D., and Hartley, B.: Crystalline Porcine Pancreatic Elastase , J Mol Biol 32, 155, 1968
 
• Shotton, D., and Hartley, B.: Evidence for the Amino Acid Sequence of Porcine Pancreatic Elastase , Biochem J 131, 643, 1973
 
• Shotton, D., and Watson, H.: Three-Dimensional Structure of Tosyl-Elastase , Nature 225, 811, 1970
 
• Stein, R., Strimpler, A., Edwards, P., Lewis, J., Mauger, R., Schwartz, J., Stein, M., Trainor, D., Wildonger, R., and Zottola, M.: Mechanism of Slow-Binding Inhibition of Human Leukocyte Elastase by Trifluoromethyl Ketones , Biochemistry 26, 2682, 1987
 
• Stein, R., Strimpler, A., Hori, H., and Powers, J.: Catalysis by Human Leukocyte Elastase: Mechanistic Insights into Specificity Requirements , Biochemistry 26, 1301, 1987
 
• Stein, R., Strimpler, A., Hori, H., and Powers, J.: Catalysis by Human Leukocyte Elastase: Proton Inventory as a Mechanistic Probe , Biochemistry 26, 1305, 1987
 
• Stein, R., Strimpler, A., Viscarello, B., Wildonger, R., Mauger, R., and Trainor, D.: Mechanism for Slow-Binding Inhibition of Human Leukocyte Elastase by Valine-Derived Benzoxazinones , Biochemistry 26, 4126, 1987
 
• Talamo, R., Levison, H., Lynch, M., Hercz, A., Hyslop, N., and Bain, H.: Symptomatic Pulmonary Emphysema in Childhood Associated with Heredity alpha-1-Antitrypsin and Elastase Inhibitor Deficiency , J Pediatrics 79, 20, 1971
 
• Thompson, R., and Blout, E.: Elastase-Catalyzed Amide Hydrolysis of Tri- and Tetrapeptide Amides , Biochemistry 12, 66, 1973
 
• Thompson, R., and Blout, E.: Peptide Chloromethyl Ketones as Irreversible Inhibitors of Elastase , Biochemistry 12, 44, 1973
 
• Tsai, Y., Juang, R., Lin, S., Chen, S., Yamasaki, M., and Tamura, G.: Production and Further Characterization of an Alkaline Elastase Produced by Alkalophilic Bacillus Strain Ya-B , Appl Environ Microbiol 54, 3156, 1988
 
• Tsai, Y., Lin, S., Lin, Y., Yamasaki, M., and Tamura, G.: Characterization of an Alkaline Elastase from Alkalophilic Bacillus Ya-B , Biochim Biophys Acta 88, 439, 1986
 
• Uram, M., and Lamy, F.: Purification of Two Proelastases from Porcine Pancreas , Biochim Biophys Acta 192, 102, 1969
 
• Vilain, A., Okochi, V., Vergely, I., Reboudravaux, M., Mazaleyrat, J., and Wakselman, M.: Acyclobenzyl Halides, Inhibitors of Elastases , Biochim Biophys Acta 1076, 401, 1991
 
• Visser, L., Sigman, D., and Blout, E.: Elastase I. A New Inhibitor, 1-Bromo-4-(2,4-dinitrophenyl) Butan-2-one , Biochemistry 10, 735, 1971
 
• Voorter, C., de Haard-Hoekman, W., Merck, K., Bloemendal, H., and de Jong, W.: Elastase Inhibition by the C-terminal Domains of alpha-Crystallin and Small Heat-shock Protein , Biochim Biophys Acta 1204, 43, 1994
 
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• Watson, H., Shotton, D., Cox, J., and Muirhead, H.: Three-Dimensional Fouriers Synthesis of Tosyl-Elastase at 3.5 Å Resolution , Nature 225, 806, 1970
 
• Williams, H., Lin, T., Navia, M., Springer, J., McKeever, B., Hoogsteen, K., and Dorn, C.: Crystallization of Human Neutrophil Elastase , J Biol Chem 262, 17178, 1987
 
• Wright, H.: Ovalbumin is an Elastase Substrate , J Biol Chem 259, 14335, 1984
 

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