Chymotrypsin
Chymotrypsin
[9004-07-3].
(kye'' moe trip' sin).
Chymotrypsin
[9004-07-3].DEFINITION
Chymotrypsin is a proteolytic enzyme crystallized from an extract of the pancreas gland of the ox, Bos taurus Linné (Fam. Bovidae). It contains NLT 1000 USP Chymotrypsin Units/mg, calculated on the dried basis, and NLT 90.0% and NMT 110.0% of the labeled potency, as determined by the Assay.
ASSAY
• Procedure
Monobasic potassium phosphate solution:
9.08 mg/mL of monobasic potassium phosphate in water
Dibasic sodium phosphate solution:
9.46 mg/mL of anhydrous dibasic sodium phosphate in water
Phosphate buffer:
Mix 38.9 mL of Monobasic potassium phosphate solution and 61.1 mL of Dibasic sodium phosphate solution. If necessary, adjust to a pH of 7.0 by the dropwise addition of Dibasic sodium phosphate solution.
Substrate solution:
Dissolve 23.7 mg of N-acetyl-l-tyrosine ethyl ester, suitable for use in assaying Chymotrypsin, in 50 mL of Phosphate buffer, with warming. When the solution is cool, dilute with additional Phosphate buffer to 100 mL. [Note—Substrate solution may be stored in the frozen state and used after thawing, but it is important to freeze it immediately after preparation. ]
Sample solution:
Dissolve a quantity of Chymotrypsin in 0.0012 N hydrochloric acid to yield a solution containing 12–16 USP Chymotrypsin Units/mL. The dilution is correct if, during the conduct of the Assay, there is a change in absorbance of between 0.008 and 0.012 in each 30-s interval.
Analysis
[Note—Determine the suitability of the substrate and check the adjustment of the spectrophotometer by performing the Analysis using USP Chymotrypsin RS in place of the Sample solution. ]
Conduct the Assay in a suitable spectrophotometer equipped to maintain a temperature of 25 ± 0.1
in the cell compartment. Determine the temperature in the reaction cell before and after the absorbance measurement to ensure that the temperature does not change by more than 0.5. Pipet 0.2 mL of 0.0012 N hydrochloric acid and 3.0 mL of Substrate solution into a 1-cm cell. Place the cell in the spectrophotometer, and adjust the instrument so that the absorbance will read 0.200 at 237 nm. Pipet 0.2 mL of Sample solution into another 1-cm cell, add 3 mL of Substrate solution, and place the cell in the spectrophotometer. [Note—Carefully follow this order of addition, and begin timing the reaction from the addition of the Substrate solution. ] Read the absorbance at 30-s intervals for NLT 5 min. Repeat the procedure on the same dilution at least once. Absolute absorbance values are less important than a constant rate of absorbance change. If the rate of change fails to remain constant for NLT 3 min, repeat the test and, if necessary, use a lower concentration. The duplicate determination of the Sample solution matches the first determination, of the same dilution, in rate of absorbance change.
in the cell compartment. Determine the temperature in the reaction cell before and after the absorbance measurement to ensure that the temperature does not change by more than 0.5. Pipet 0.2 mL of 0.0012 N hydrochloric acid and 3.0 mL of Substrate solution into a 1-cm cell. Place the cell in the spectrophotometer, and adjust the instrument so that the absorbance will read 0.200 at 237 nm. Pipet 0.2 mL of Sample solution into another 1-cm cell, add 3 mL of Substrate solution, and place the cell in the spectrophotometer. [Note—Carefully follow this order of addition, and begin timing the reaction from the addition of the Substrate solution. ] Read the absorbance at 30-s intervals for NLT 5 min. Repeat the procedure on the same dilution at least once. Absolute absorbance values are less important than a constant rate of absorbance change. If the rate of change fails to remain constant for NLT 3 min, repeat the test and, if necessary, use a lower concentration. The duplicate determination of the Sample solution matches the first determination, of the same dilution, in rate of absorbance change. Determine the average absorbance change per min, using only the values within the 3-min portion of the curve where the rate of absorbance change is constant. Plot a curve of absorbance against time. One USP Chymotrypsin Unit is the activity causing a change in absorbance of 0.0075/min under the conditions specified in the Assay.
Calculate the number of USP Chymotrypsin Units/mg in the portion of Chymotrypsin taken:
Result = (A2 
A1)/(T × W × F)
A1)/(T × W × F)| A2 | = | = absorbance straight-line initial reading |
| A1 | = | = absorbance straight-line final reading |
| T | = | = time elapsed between the initial and final readings (min) |
| W | = | = weight of Chymotrypsin in the volume of solution used in determining the absorbance (mg) |
| F | = | = Chymotrypsin activity conversion factor, 0.0075/min |
Acceptance criteria:
NLT 1000 USP Chymotrypsin Units/mg on the dried basis; 90.0%–110.0% of the labeled potency
IMPURITIES
• Residue on Ignition 
281
:
NMT 2.5%
281:
NMT 2.5%
• Limit of Trypsin
Tris buffer:
Dissolve 294 mg of calcium chloride in 40 mL of 0.20 M tris(hydroxymethyl)aminomethane. Adjust with 1 N hydrochloric acid to a pH of 8.1, and dilute with water to 100 mL.
Substrate solution:
Transfer 98.5 mg of p-toluenesulfonyl-l-arginine methyl ester hydrochloride, suitable for use in assaying trypsin, to a 25-mL volumetric flask. Add 5 mL of Tris buffer, and swirl until the substrate dissolves. Add 0.25 mL of methyl red–methylene blue TS, and dilute with water to volume.
Sample solution:
10 mg/mL of Chymotrypsin in water
Analysis
[Note—Determine the suitability of the substrate by performing the Analysis using the appropriate amount of USP Trypsin Crystallized RS in place of the Sample solution. ]
By means of a micropipet, transfer 50 µL of Sample solution to a depression on a white spot plate. Add 0.2 mL of Substrate solution.
Acceptance criteria:
No purple color develops within 3 min (NMT 1% of trypsin).
SPECIFIC TESTS
• Microbial Enumeration Tests 61 and Tests for Specified Microorganisms 62:
It meets the requirements of the tests for absence of Pseudomonas aeruginosa, Salmonella species, and Staphylococcus aureus.
61 and Tests for Specified Microorganisms 62:
It meets the requirements of the tests for absence of Pseudomonas aeruginosa, Salmonella species, and Staphylococcus aureus.
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight containers, and avoid exposure to excessive heat.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Krishna Menon
Associate Director, Biologics +91 40 44488762 |
(BIO12010) Monographs - Biologics and Biotechnology 1 |
| 61 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison (301) 816-8339 |
(GCM2010) General Chapters - Microbiology |
| 62 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison (301) 816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP38–NF33 Page 2801