细胞定位：Secreted.
序列相似性：Belongs to the peptidase A1 family.
Pepsin hydrolyzes peptide bonds, not amide or ester linkages. Pepsin cleaves peptides with an aromatic acid on either side of the peptide bond. Sulfur-containing amino acids increase susceptibility to hydrolysis when they are close to the peptide bond. Pepsin preferentially cleaves at the carboxyl side of phenylalanine and leucine and at the carboxyl side of glutamic acid residues. Cleaves Phe-Val, Gln-His, Glu-Ala, Ala-Leu, Leu-Tyr, Tyr-Leu, Gly-Phe, Phe-Phe and Phe-Tyr bonds in the β chain of insulin.
Pepsin is the major proteolytic enzyme produced in the stomach. It digests proteins through the cleavage of interior peptide linkages.
Preferential cleavage: hydrophobic and aromatic residues in P1 and P1′ postitions. Cleaves Phe-Val, Gln-His, Glu-Ala, Ala-Leu, Leu-Tyr, Tyr-Leu, Gly-Phe, Phe-Phe and Phe-Tyr bonds in the β chain of insulin.
Pepsin Activity
Application: This procedure is used to determine the protease activity of pepsin preparations derived from porcine or other animal stomach tissue.
Reagents and Solutions
1. Dilute hydrochloric acid solution: Dilute 30 mL of 1.0 N hydrochloric acid with water to 1000 mL, then adjust to a pH of 1.6 ± 0.1 with 1.0 N hydrochloric acid, if necessary.
2. TCA solution: Prepare a 4.0% (w/v) solution of trichloroacetic acid in water.
3. Substrate solution: Weigh 5.0 g of USP Hemoglobin Protease Substrate RS into a large beaker. Add 100 mL of Dilute hydrochloric acid solution to the beaker, and stir using a magnetic stirrer. Once the hemoglobin is fully dissolved, adjust the pH of the solution to 1.6 ± 0.1 (use 1.0 N hydrochloric acid, dropwise, to adjust the pH as needed). Quantitatively transfer the solution to a 250-mL volumetric flask, then dilute with Dilute hydrochloric acid solution to volume. [Note—Prepare fresh daily.]
4. Standard solutions: Quantitatively prepare four serial dilutions of USP Pepsin for Assay RS in Dilute hydrochloric acid solution covering the activity range of 0.70–1.25 USP Pepsin U/mL. [Note—Prepare immediately before use.]
5. Sample solution: Quantitatively prepare a solution of pepsin in Dilute hydrochloric acid solution containing approximately 0.9–1.0 USP Pepsin U/mL. [Note—Prepare immediately before use.]
Procedure
1. For the standard curve, prepare test tubes as follows (use tubes that can accommodate and allow vortex mixing of a volume of NLT 16 mL). Separately transfer 1.0-mL aliquots of each of the Standard solutions into four test tubes—two tubes for the analysis of each Standard solution and two blank tubes for each Standard solution. Prepare a substrate blank by transferring 1.0 mL of Dilute hydrochloric acid solution into a single test tube. A full set of tubes for creation of the standard curve will require seventeen test tubes.
2. For each pepsin sample being tested, prepare test tubes as follows. Separately transfer 1.0-mL aliquots of the Sample solution into four test tubes—two tubes for the analysis of each sample and two blank tubes for each sample.
3. To each of the blank tubes (the substrate blank, the Standard solutions blanks, and the Sample solution blanks), add 10.0 mL of the TCA Solution, and mix by vortexing. Place all of the test tubes into a water bath maintained at 25° ± 0.1°. Add 5.0 mL of the Substrate solution (previously equilibrated to 25° ± 0.1°) to each of the blank tubes, and mix by vortexing. Using a stopwatch and starting at time equals zero, rapidly pipet 5.0 mL of the Substrate solution (previously equilibrated to 25° ± 0.1°) successively and at intervals of exactly 30 s into each of the tubes containing the Sample solution and Standard solutions for analysis. Vortex each tube immediately after adding the Substrate solution, then return the tube to the water bath. Exactly 10.0 min after the addition of the Substrate solution, stop the reaction by rapidly pipetting 10.0 mL of the TCA solution into each analysis tube at intervals of exactly 30 s, immediately vortexing each tube after addition of the TCA solution. Remove all tubes from the water bath.
4. Allow all of the tubes (including blanks) to sit at room temperature for 25 min. Prepare clean test tubes for filtering the solutions, allowing two tubes for each solution to be filtered. Place funnels in each tube, and line the funnels with fluted or folded filter paper. After 25 min, vortex each tube, then transfer the contents of each tube into a clean test tube. Refilter each of the filtrates through the same paper into a second clean test tube. Measure the absorbance of each filtrate at 280 nm using a suitable UV-visible spectrophotometer that has previously been zeroed with the substrate blank in a 1-cm quartz cell. Record the absorbance reading for each Standard solution, Sample solution, and their respective blanks.
Determine the net absorbance for each of the Standard solutions by subtracting the average absorbance of the Standard solution blank from the average absorbance of the corresponding Standard solution. Plot a standard curve of the net absorbance of each Standard solution versus its concentration, in mg/mL. Determine the slope (m) and y-intercept (b) of the resulting curve.
Calculation
One pepsin unit is defined as that quantity of enzyme that produces the equivalent of 1 μmol of tyrosine per min under the conditions of the assay.
Calculate the activity of the enzyme preparation by the equation:
in which：
As: The average absorbance for the sample corrected for the average absorbance of the sample blanks;
b: The y-intercept of the standard curve;
P: The activity of the USP Pepsin for Assay RS used to prepare the Standard solutions (U/mg);
m: The slope of the standard curve;
C: The concentration of the Sample solution (mg/mL).
D2601380
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