Data Sheet - Trypsin Chymotrypsin Mixture

Source:
Bovine or porcine pancreas
Systematic name:
Peptidyl peptide hydrolase

Chymotrypsin

Occurrence

Chymotrypsin is a proteolytic enzyme which has been isolated from many vertebrates. It is synthesized in the pancreas in the form of an inactive precursor or zymogen, named Chymotrypsinogen. This precursor is transformed into the active enzyme by Trypsin and by Chymotrypsin itself.

Characteristics

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

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 a1-antichymotrypsin, a1- proteinase inhibitor (formerly: a1-antitrypsin), a2-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 Aa 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)

Trypsin

Occurrence

Trypsin is a proteolytic enzyme found in many animals and even bacteria. In vertebrates an inactive precursor, the Trypsinogen, is formed in the pancreas. By the action of Enterokinase or of Trypsin itself, Trypsinogen is transformed into active Trypsin.

Characteristics

Specificity: Trypsin hydrolyzes peptides, amides and esters at bonds involving the carboxyl group of L-arginine and L-lysine. Trypsin acts on numerous synthetic substrates, the best being esters of L-arginine, e.g. N-benzoyl-L-arginine ethyl ester. Proteins are only slowly degraded unless denatured (1).

Effectors: In the process of Trypsinogen activation Ca2+ ions have an accelerating effect and they are also essential for complete activation (1,2,3).

Trypsin is inhibited by a wide variety of substances. Trypsin is a serine proteinase (EC 3.4.21) and is therefore inhibited by compounds such as phenylmethyl-sulfonylfluoride (PMSF) and diisopropylphosphofluoridate (DFP). Aromatic and aliphatic amidines and amines are also inhibitory, the strongest low molecular weight competitive inhibitor of Trypsin being p-aminobenzamidine (1,4). Tosyl-L-lysine chloromethyl ketone (TLCK) irreversibly inhibits Trypsin (but not Chymotrypsin) (1,2,5).

High molecular weight inhibitors of Trypsin comprise polypeptides and proteins from animal origin (aprotinin, α2-macroglobulin, a1-antitrypsin, bovine pancreatic Trypsin inhibitor etc., as well as those isolated from plants e.g. from soybean, lima bean, and barley (see also: Methods in Enzymology, Vol. XIX, pp. 840-915).

Catalytic optima: Trypsin acts optimally at pH values between 7 and 9 (1,6).

Stability: Trypsin is most stable at pH 3. At this pH at low temperature it retains its activity for weeks (1,2). Autolysis of Trypsin at pH values well above 3 can be retarded by the addition of calcium ions. This protective effect is much more pronounced with bovine Trypsin than with porcine Trypsin (2). Heat denaturation is dependent on the pH: below pH 8 an increased temperature leads to reversible denaturation, but above pH 8 a temperature rise induces irreversible denaturation (2,3). Precipitation with trichloroacetic acid or the presence of high concentrations of urea (8 M) leads to a reversible loss of Trypsin activity (1). Lyophilized Trypsin, when stored cool and dry in the dark, is stable almost indefinitely (7).

Solubility: Trypsin is soluble in water and in isotonic saline solution.

Molecular weight: approx. 23,500 (4).

Composition: Bovine Trypsinogen consists of a single polypeptide chain with 229 amino acid residues stabilized by 6 disulfide bridges. Upon activation, a hexapeptide (Val-(Asp)4-Lys-) is split off from the amino terminus leading to the formation of β-Trypsin, the first active enzyme form. Sequential cleavage at two other sites leads to α-Trypsin, and Pseudotrypsin, respectively, both of which are also active forms of Trypsin (1,2,3).

Isoelectric point: 10.8 (bovine and porcine Trypsin) (1,3).

Spectral data: E280 (1%, 1cm) = 15.4 (1).

Assay (Activity of Chymotrypsin according to current 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.

    For 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.
    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

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 (= Δ 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 „Δ A/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.

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

Assay (Activity of Trypsin according to current USP-Monograph)

Definition:
One USP Trypsin Unit is the activity which hydrolses 1 µmol Na- Benzoyl-L-argininethylesterhydrochlorid (= BAEE*HCl) such that causing a change in absorbance of 0.003 per minute under the conditions specified in this assay at 253 nm.

Reagents

  1. Temperature equalisation: Trypsin Reference Standard USP, the Samples and BAEE*HCl (= Na- Benzoyl-L-argininethylesterhydrochlorid) from 4 °C to Room temperature.
  2. 1 mM HCl-Solution: 1.0 ml 1 N HCl-Solution + pure H2O add. to 1000 ml.
  3. BAEE-Solution: 85.7 mg BAEE*HCl (M = 342.83 g/mol, Calbiochem 2645-08-1) + pure H2O add to 100.0 ml.
  4. 0.067 M KH2PO4-Solution: 4.56 g KH2PO4 (M=136.09 g/mol, MERCK 4873) + pure H2O add to 500 ml.
  5. 0.067 M Na2HPO4-Solution: 4,73 g Na2HPO4 (M = 141,96 g/mol, MERCK 6586) + pure H2O add to 500 ml.
  6. 0.067 M Phosphat-Buffer, pH 7.60: Give to 200 ml Na2HPO4-Solution an amount of KH2PO4-Solution until it adjust a pH of 7.60.
  7. Enzyme-Solution: Test the Activity of each Sample with two Initial weight.
    For 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.
    Weight an amount of Enzyme Sample or Standard that solved and diluted in 1 mM HCl-Solution give a change of absorbance (=Δ A/min) of maximal 0.02 in „ml in Test“ of 0.200.

 


Initial weight (mg) =
[(5.0-10.0 USP-U/“0.200“ ml in Test)*dilution factor*Enzyme solution]
Declared activity of enzyme

Procedure

For each 90 ml of Phosphat-Buffer use 10 ml BAEE-Solution (1:10). The Absorbance should be at 253 nm 0.575 - 0.585. If necessary add more Phosphat-Buffer or BAEE-Solution.
Put the amount Phosphate-Buffer-Solution in Bath thermostat to adjust the temperature to 25 °C. Shortly before test begin give the necessary amount of BAEE-Solution. The Solution at 25 °C is stable for 2 h. To reach a homogeny temperature, mix the solution before using each time.

  1. Adjust the UV-Spectral photometer such that the change of absorbance for each reaction in the cuvettes can be reading every 30 seconds for 5 minutes.
  2. Calibrate the UV-Spectral photometer at 253 nm using pure H2O.
  3. Blank: Give in a cuvette 200 µl of 1 mM HCl-Solution and 3 ml of Phosphate-Buffer-Solution and mix. Calibrate the UV-Spectral photometer with this solution.
  4. After calibration give in the cuvette e.g. 100 µl of Enzyme dilution, 100 µl of 1 mM HCl-Solution and 3 ml of Phosphate-Buffer-Solution. Mix immediately put in UV-Spectral photometer and start reading the absorbance.

    Is the Change of Absorbance (= Δ A/min) higher than 0.02 or lower than 0.009 then change for the next measurement the amount of “µl in Test” of 80-200 µl.

  5. Print the << Rates >> of absorbance and the tabulate of „Δ A/min“.
  6. Prepare the UV-Spectral photometer according the manufacturer instruction to starting a new measurement.
  7. Measure each Enzyme dilution 4 times.
  8. Clear the cuvette for each using with pure H2O.

Calculation

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


Factor =
                                                                    1                                                             
{[(Initial weight in mg/Enzyme Solution in ml)/(Dilution factor)]*(ml in Test)} * 0.003

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

Chymotrypsin 1000/1000
Trypsin 2400:400
Trypsin 900:150
Trypsin 1200:300
Trypsin 800:330

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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