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Enzyme Explorer

Pepsin

E.C. 3.4.23.1

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Physical Properties

Pepsin, a member of the Peptidase A1 family, is the predominant digestive protease in the gastric juice of vertebrates.
Molecular Weight: 34,620 (from porcine amino acid sequence)1
pI: 2.2 - 3.02; 2.2, 2.83 (porcine)
λmax: 278 nm4 Extinction coefficient: EmM = 51.34 (porcine)

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Specificity

Pepsin, unlike some other endopeptidases, hydrolyzes only peptide bonds. It does not hydrolyze non-peptide amide or ester linkages.

Pepsin exhibits preferential cleavage for hydrophobic, preferably aromatic, residues in P1 and P1' positions. Increased susceptibility to hydrolysis occurs if there is a sulfur-containing amino acid close to the peptide bond, which has an aromatic amino acid.

Cleaves Phe1Val, Gln4His, Glu13Ala, Ala14Leu, Leu15Tyr, Tyr16Leu, Gly23Phe, Phe24Phe and Phe25Tyr bonds in the B chain of insulin.5

Pepsin will also preferentially cleave at the carboxyl side of phenylalanine and leucine and to a lesser extent at the carboxyl side of glutamic acid residues. Pepsin will not cleave at valine, alanine, or glycine linkages.6 Amidation of the C-terminal carboxyl group prevents hydrolysis by pepsin.6,7

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Applications

Pepsin is commonly used in the preparation of F(ab')2 fragments from antibodies. In some assays it is preferable to use only the antigen binding (Fab) portion of the antibody. For these applications, antibodies may be enzymatically digested to produce either an Fab or an F(ab')2 fragment of the antibody. To produce an F(ab')2 fragment, IgG is digested with pepsin, which cleaves the heavy chains near the hinge region. One or more of the disulfide bonds that join the heavy chains in the hinge region are preserved, so the two Fab regions of the antibody remain joined together, yielding a divalent molecule (containing two antibody binding sites), hence the designation F(ab')2. The light chains remain intact and attached to the heavy chain. The Fc fragment is digested into small peptides. Fab fragments are generated by cleavage of IgG with papain instead of pepsin. Papain cleaves IgG above the hinge region containing the disulfide bonds that join the heavy chains, but below the site of the disulfide bond between the light chain and heavy chain. This generates two separate monovalent (containing a single antibody binding site) Fab fragments and an intact Fc fragment. The fragments can be purified by gel filtration, ion exchange, or affinity chromatography. Protocols for antibody digestion and purification of antibody fragments can be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988 (A2926).

Fab and F(ab')2 antibody fragments are used in assay systems where the presence of the Fc region may cause problems. In tissues such as lymph nodes or spleen, or in peripheral blood preparations, cells with Fc receptors (macrophages, monocytes, B lymphocytes, and natural killer cells) are present which can bind the Fc region of intact antibodies, causing background staining in areas that do not contain the target antigen. Use of F(ab')2 or Fab fragments ensures that the antibodies are binding to the antigen and not Fc receptors. These fragments may also be desirable for staining cell preparations in the presence of plasma, because they are not able to bind complement, which could lyse the cells. F(ab')2, and to a greater extent Fab, fragments allow more exact localization of the target antigen, i.e. in staining tissue for electron microscopy. The divalency of the F(ab')2 fragment enables it to cross-link antigens, allowing use for precipitation assays, cellular aggregation via surface antigens, or rosetting assays. The optimal pH for the pepsin reaction is 1.5-2.5, which will not be detrimental to the antibody, if it is not exposed for long durations to the low pH. Solutions should be adjusted to neutral pH for storage. The control of pepsin digestion of antibodies has been reported.8


Pepsin Cleavage


For general digestion of proteins, suggested conditions are a 0.4% solution of pepsin in 10 mM HCl, and digestion for 30-90 minutes at 37 °C. Pepsin has optimal activity with native proteins at approximately pH 1.0, but with some denatured proteins the optimal activity is at approximately pH 1.5-3.5.9,10

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Inhibitors
Product No. Product Name Add to
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P5318 Pepstatin A microbial, ≥90% (HPLC)  

P7424 Pepsinostreptin ≥97% (HPLC)
P7626 Phenylmethanesulfonyl fluoride ≥98.5% (GC)

Substrates
Product No. Product Name Add to
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H2625 Hemoglobin from bovine blood suitable for protease substrate, substrate powder
F5255 Fibrin Blue suitable for substrate for pepsin at low pH (High blanks result at high pH)
96150 Z-Glu-Tyr BioChemika, substrate for the det. of pepsin, ≥99.0% (HPLC)
77431 Phe-Ala-Ala-Phe(4-NO2)-Phe-Val-Leu (4-pyridylmethyl) ester BioChemika, ≥98.0% (TLC)

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Kinetics, Solubility and Solution Stability

Pepsin is soluble in deionized water at 1% (10 mg/ml) and at 0.4% (4 mg/ml) in cold 10 mM hydrochloric acid. Solutions at pH 4.4 are stable at -20 °C for about 2-3 months.12 The pH optimum for activity for porcine pepsin is ~2.2. At pH 1.5 pepsin exhibits about 90% of maximum activity, and at pH 4.5 about 35% of maximum activity.13 Solutions are stable at pH 6-7. Bringing the pH up to 8; however, will irreversibly inactivate pepsin. Pepsin is irreversibly denatured at pH 8.5 - 11 at room temperature.14



Products
Product No. Product Name Add to
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P7000 Pepsin from porcine gastric mucosa, powder, ≥250 units/mg solid
P6887 Pepsin from porcine gastric mucosa lyophilized powder, 3,200-4,500 units/mg protein
P7012 Pepsin from porcine gastric mucosa, lyophilized powder, ≥2,500 units/mg protein
P7125 Pepsin from porcine gastric mucosa, powder, ≥400 units/mg protein
P0609 Pepsin−Agarose from porcine gastric mucosa lyophilized powder, 30-70 units/mg dry solid
P1490 Pepsinogen I human
P4656 Pepsinogen from porcine stomach Grade I-S, lyophilized powder, ~3,000 units/mg protein (after activation to pepsin at pH 2.0 at 25°C)

References

  1. Sepulveda. P., et al., Primary Structure of Porcine Pepsin. III. Amino Acid Sequence of a Cyanogen Bromide Fragment, CB2A, and the Complete Structure of Porcine Pepsin. J. Biol. Chem., 250, 5082 (1975).
  2. Jonsson, M., Isoelectric Spectra of Native and Base Denatured Crystallized Swine Pepsin. Acta Chem. Scand., 26, 3435-3440 (1972).
  3. Malamud, D., and Drysdale, J.W., Isoelectric Points of Proteins: A Table, Anal. Biochem., 86, 620-647 (1978).
  4. Proc. Natl. Acad. Sci., 45, 915-922 (1959).
  5. IUBMB Enzyme Nomenclature: http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/23/1.html
  6. Sweeney, P.J., and Walker, J.M., in Enzymes of Molecular Biology, Burrell, M.M., ed., Humana Press (Totowa, NJ: 1993), pp. 290-291.
  7. Enzymes, Dixon, M., et al., Academic Press (New York, NY: 1979), p. 262.
  8. Rea, D.W., and Ultee, M.E., A Novel Method for Controlling the Pepsin Digestion of Antibodies. J. Immunol. Meth., 157, 165-173 (1993).
  9. Arch. Biochem. Biophys., 57, 163-173 (1955).
  10. J. Biol. Chem., 234, 3137-3145 (1959).
  11. Knowles, J.R., et al., The pH-dependence of the Binding of Competitive Inhibitors to Pepsin. Biochem. J., 113, 343-51 (1969).
  12. Rajagopalan, T.G., et al., Pepsin from Pepsinogen. Preparation and Properties. J. Biol. Chem., 241, 4940 (1966).
  13. Bohak, Z.; J. Biol. Chem,. 244, 4638-4648 (1969)
  14. Ryle, A.P., The Porcine Pepsins and Pepsinogens. Methods in Enzymol., 19, 316-336 (1970).

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