Data Sheet - Chymotrypsin

Source:
Bovine or porcine pancreas
Systematic name:
Peptidyl peptide hydrolase
CAS-No.
9004-07-3
E.C.-No.
3.4.21.1
EG-Indexnummer
647-011-00-2

Occurrence

Unless otherwise specified, this paragraph will deal with literature data on bovine Chymotrypsin Aα

Characteristics

Specificity:Chymotrypsin hydrolyzes peptides, amides and esters at bonds involving the carboxyl group of L-tyrosine, L-tryptophan and L-phenylalanine. Proteolysis also occurs, although much slower, adjacent to other large hydrophobic amino acid residues, such as L-leucine, L-methionine and L-histidine (1,2). In assays for Chymotrypsin activity the synthetic substrates N-acetyl-L-tyrosine ethyl ester, N-benzoyl-L-tyrosine ethyl ester and N-acetyl-L-tryptophan amide are most frequently used (3).

Effectors:Ca2+ ions have been shown to enhance the activity of Chymotrypsin and also to stabilize the enzyme against denaturation (3,4).

Chymotrypsin is inhibited by several low molecular weight and high molecular weight substances. Indole, b-indolepropionate, p-iodo-phenylacetate as well as D stereoisomers of substrates, e.g. N-acetyl-D-tyrosine ethyl ester or N-acetyl-D-tryptophan methyl ester, behave as competitive inhibitors (1). Like other serine proteases, Chymotrypsin is inhibited by phenylmethylsulfonylfluoride (PMSF) and diisopropylphosphofluoridate (DFP) (1,5). Tosylphenylalanylchloromethane (TPCK) irreversibly inhibits Chymotrypsin (but not Trypsin) (3). Heavy metals like Cu2+ and Hg2+ are also inhibitory (3,4). Among the most important high molecular weight inhibitors are proteins like α1-antichymotrypsin, α1- proteinase inhibitor (formerly: a1-antitrypsin), α2-macroglobulin, aprotinin (pancreatic Trypsin inhibitor, Kunitz inhibitor), soybean Trypsin inhibitor and Ascaris inhibitors (3,5; see also: Methods in Enzymology, Vol. XIX, pp. 844-905).

Catalytic optima:Chymotrypsin acts optimally at pH values around 8 (6).

Stability:As a lyophilized powder, Chymotrypsin is stable almost indefinitely provided it is stored dry in a cool place (7). In solution Chymotrypsin is most stable at pH 3. Below pH 3 the enzyme is reversibly denatured. Above pH 10 Chymotrypsin becomes inactive. The enzyme is stabilized by Ca2+ ions (3, 4).

Solubility:Chymotrypsin is sparingly soluble in water.

Molecular weight:approx. 25,000 (2,3,6).

Composition:Chymotrypsin Aa consists of 241 amino acid residues in 3 poly-peptide chains, A (13 residues), B (131 residues) and C (97 residues), which are held together by 5 disulfide bridges (3,4). Chymotrypsin Aα arises from the inactive single-chain precursor Chymotrypsinogen A by the successive cleavage of 4 peptide bonds with a concomitant removal of 2 dipeptides, Ser14-Arg15 and Thr147-Asn148. Only one of the 4 cleavages is catalyzed by Trypsin (Arg15-Ile16) and it is basically this one which renders Chymotrypsinogen active; the other 3 are autolytic cleavages by Chymotrypsin (2,3,4,8). The activation of Chymotrypsinogen A to Chymotrypsin Aa proceeds via various intermediate forms. The amount of Trypsin present during the activation governs the appearance either of several active interme-diate forms (Chymotrypsin Api, Adelta, Akappa, Agamma) or of inactive forms, called neo-chymotrypsinogens (3,4,8).

Isoelectric point: approx. 8.4 (4).

Spectral data: E282 (1%, 1cm) = 20.0 (4,6).

Assay (according to current FIP-Method)

The method described here is the one given in the monograph on Chymotrypsin in the Ph.Eur. It is identical to the method given by FIP (Fédération International Pharmaceutique; see 3) which originally developed this assay method.

Principle
Chymotrypsin hydrolyzes the synthetic substrate N-acetyl-L-tyrosine ethyl ester (ATEE). The amount of acid liberated (at pH 8.0 and 25 °C) is measured by titration with sodium hydroxide, recorded as a function of time.
The activity of Chymotrypsin is determined by comparing the rate at which it hydrolyzes acetyltyrosine ethyl ester with the rate at which Chymotrypsin BRP hydrolyzes the same substrate under the same conditions.

Apparatus
Use a reaction vessel of about 30 ml capacity provided with:

An automatic or manual titration apparatus may be used. For the latter, the burette is graduated in 0.005 ml and the pH meter is provided with a wide scale and glass-calomel electrodes.

Reagents

  1. ATEE solution (0.2 M): Dissolve 0.539 g of N-acetyl-L-tyrosine ethyl ester monohydrate (ATEE x H2O) in ethanol 96% and dilute to 10.0 ml with the same solvent.
  2. Calcium chloride solution (0.01 M): Dissolve 0.147 g of CaCl2 x 2H2O in water and dilute to 100.0 ml with the same solvent.
  3. 0.02 N Sodium hydroxide.
  4. 0.001 N Hydrochloric acid.
  5. Test solution: Dissolve 25.0 mg of the substance to be examined in 0.001 N hydrochloric acid (D) and dilute to 250.0 ml with the same acid.
  6. Reference solution: Dissolve 25.0 mg of Chymotrypsin BRP in 0.001 N hydrochloric acid (D) and dilute to 250.0 ml with the same acid

Chymotrypsin BRP is issued by: Technical Secretariat, European Pharmacopoeia Commission, Council of Europe, P.O. Box 907, 67029 Strasbourg CEDEX 1, France.

Store the test solution (E) and the reference solution (F) at 0 °C to 5 °C. Warm 1 ml of each solution to about 25 °C over 15 min and use 50 ml of each solution (corresponding to about 25 nanokatals) for each titration. Carry out the titration in an atmosphere of nitrogen.

Procedures

Transfer into the reaction vessel 10.0 ml of calcium chloride solution (0.01 M) (B) and add, while stirring 0.35 ml of ATEE solution (0.2 M) (A). When the temperature is steady at 25.0 ± 0.1 °C, adjust the pH to exactly 8.0 with 0.02 N sodium hydroxide (C), then add 50 µl of test solution (E), equivalent to 5 µg of substance, start stop-watch and maintain the pH at 8.0 by the addition of 0.02 N sodium hydroxide (C). Note the volume added every 30 s.

Calculate the volume of 0.02 N sodium hydroxide used per second between 30 s and 210 s. Carry out a titration in the same manner using the reference solution and calculate the volume of 0.02 N sodium hydroxide used per second.

Calculate the activity in microkatals per milligram using the expression:

m´ x V
x A
m x V´

m  = mass in milligrams of the substance to be examined,
m´= mass in milligrams of Chymotrypsin BRP,
V = volume of 0.02 N sodium hydroxide used per second by the test solution,
V´= volume of 0.02 N sodium hydroxide used per second by the reference solution,
A = activity of Chymotrypsin BRP in microkatals per milligram.

Other assays

In the FIP assay for Chymotrypsin (see above) the specific activity is given in FIP units/mg. 1 µkatal/mg = 60 FIP units/mg.

In the assay for Chymotrypsin activity according to USP 24 the rate of hydrolysis of ATEE at pH 7.0 is measured spectrophotometrically at 237 nm.

Assay (according to USP-Monograph)

Definition
One USP-Unit of Chymotrypsin correspond the amount of Enzyme, that under the test conditions hydrolysed 1 µmol N-Acetyl-L-tyrosinethylester (= ATEE), that makes a Change of Absorbance of 0.0075 pro Minute at 237 nm.

Preparation of solutions

  1. Temperature equalisation: Chymotrypsin Reference Standard USP, the Samples and N-Acetyl-L-tyrosinethylester from 4 °C to Room temperature.
  2. 1 mM HCl-Solution: 1.0 ml 1 N HCl-Solution + pure H2O add 1000 ml.
  3. N-Acetyl-L-tyrosinethylester-Solution: 61.0 mg ATEE*H2O (M = 269.30 g/mol) + 96%ige EtOH add to 1.0 ml.
  4. 0.067 M KH2PO4-Solution: 4.56 g KH2PO4(M=136.09 g/mol, MERCK 4873) + pure H2O add 500 ml.
  5. 0.067 M Na2HPO4-Solution: 4,73 g Na2HPO4 (M = 141,96 g/mol, MERCK 6586) + pure H2O add 500 ml.
  6. 0.067 M Phosphat-Buffer, pH 7.00: Give to 200 ml KH2PO4-Solution the amount of Na2HPO4-Solution until the pH-value adjust to 7.00.
  7. Enzyme-Solution: Test the Activity of each Sample with two Initial weight. Weight the amount of Enzyme Sample or the Standard and solve in 1 mM HCl-Solution. The Dilution of this Enzyme soluion in 1.2 mM HCl-Solution give a change of absorbance (= ΔA/min) of maximal 0.02 in „ml in Test“ of 0.200.

Formula:

mg Initial weight =      [(1.9 USP-U/„0.200 ml/Test“)* dilution factor*Enzyme-Solution] / declared Activity of Enzyme

If the Sample is a Trypsin- und Chymotrypsin-Mixture, make an initial weight of the Sample that the Try- and Chy-Activity can be determined according to USP from the same Enzyme Solution (50 ml) with a „ml in Test“ of 0.080-0.200 ml.

Procedure

Give in 100 ml Phosphat-Buffer 0.42 ml of ethanolic ATEE-Solution. Put Phosphat-Buffer-Solution in Bath thermostat to adjust the temperature to 25 °C. Shortly before to beginning with the test give the ATEE-Solution into buffer. This Solution is stabile at 25 °C for about 1.5 h. To reach a homogeny temperature, mix the solution before using each time

  1. Calibrate the UV-spectral photometer at 237 nm with pure H2O.
  2. For each test measure a Blank (200 µl of 1.2 mM HCl-Solution). Calibrate the UV-Spectral photometer with this Blank.
  3. For testing of Enzyme-solution: E.g. give in a cuvette 80 µl of Enzyme-dilution and 120 µl of 1.2 mM HCl-solution.
  4. Mix the Buffer-ATEE-Solution each time before using. Give into this cuvette 3 ml of ATEE-Buffer-Solution, mix and put it immediately in spectral photometer and start the measurement.
  5. Measure the absorbance of the reaction every 30 seconds for 5 Minutes.Is the Change of Absorbance (= D A/min) higher than 0.02 or lower than 0.009 then change for the next measurement the amount of “µl in Test” 80 - 200 µl.
  6. Print the << Rates >> of absorbance and the tabulate of „ΔA/min“.
  7. Prepare the UV-Spectral photometer according the manufacturer instruction to starting a new measurement.
  8. Measure each Enzyme dilution 4 times.
  9. Clear the cuvette for each using with pure H2O.

Calculation:

Calculate the average of D A/min of each Enzyme-Solution.


Factor =
                                                                     1                                                                 
{[(Initial weight in mg/Enzyme Solution in ml)/(Dilution factor)] * (ml in Test)} * 0.0075

USP-unit/mg (abs.) = Δ A/min * Factor
Standard: USP-unit/mg (rel.) = r = USP-unit/mg (abs.) / declared Activity of reference Standard
Sample: USP-unit/mg (rel.) = USP-unit/mg (abs.) / r

Availability

Standard qualities
Chymotrypsin 1400 USP units/mg
Chymotrypsin 1000 USP units/mg
Chymotrypsin  Ph. Eur. (5 mkatal/mg)
Chymotrypsin 300 FIP units/mg

Customized qualities are available upon request.

References

  1. Blow, D.M. in: The Enzymes (P.D. Boyer, ed.) 3rd ed., Vol. III, p.185. Academic Press, New York, 1971.
  2. Hess, G.P. in: The Enzymes (P.D. Boyer, ed.) 3rd ed., Vol. III, p.213. Academic Press, New York, 1971.
  3. Lauwers, A., Scharpé, S.: Pharmaceutical Enzymes. drugs and pharmaceutical sciences., Volume 84, Marcel Dekker, Inc., New York-Basel-Hong Kong, 1997.
  4. Wilcox, P.E. in: Methods in Enzymology (G.E. Perlmann, L. Lorand, eds.) Vol. XIX, p.64. Academic Press, Inc., Orlando, Florida, 1970.
  5. Geiger, R. in: Methods of Enzymatic Analysis (Bergmeyer, J., Graál, M., eds.) 3rd ed., Vol. V, p.99. Verlag Chemie, Weinheim, 1984.
  6. Walsh, K.A., Wilcox, P.E. in: Methods in Enzymology (G.E. Perlmann, L. Lorand, eds.) Vol. XIX, p.31. Academic Press, Inc., Orlando, Florida, 1970.
  7. Stellmach, B.: Bestimmungsmethoden Enzyme. Steinkopff Verlag Darmstadt, 1988.
  8. Kraut, J. in: The Enzymes (P.D. Boyer, ed.) 3rd ed., Vol. III, p.165. Academic Press, New York, 1971.

 

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