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).

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

Extinction Coefficient

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

Isoelectric

9.5 (Bieth 2004)

CAS Registry Number

C.A.S.: 9004-06-2

Technical Notes

1 SucAla3NA unit is approximately equivalent to 6 elastin digestion units. Aqueous liquid suspensions should be aseptically handled to avoid bacterial contamination. Due to the viscous nature of the aqueous suspension the vial should be rinsed to recover contents.

Storage Conditions

Elastase is unstable at pH ≤ 3.5. When stored as a dry powder the enzyme is stable for 6-12 months at 2-8°C. It is helpful to make primary solutions in KCl or alkaline buffers before diluting into the reaction mixtures or media, compensating for ionic strength or pH changes. Stable at pH 4.0-10.4.