Trypsin, a kind of protease, EC 3.4.4.4, is a serine proteolytic enzyme extracted from the pancreas of cattle, sheep and pigs.In vertebrates, it functions as a digestive enzyme.After the pancreas is the precursor of trypsin, trypsinogen is synthesized and secreted as a component of pancreatic juice. It is restricted by intestinal kinase or trypsin and can be decomposed into activated trypsin, which is an endopeptidase. It can cut the carboxyl side of lysine and arginine residues in the polypeptide chain.It not only acts as a digestive enzyme, but also can restrict the decomposition of chymotrypsinogen, carboxypeptidase, phospholipase and other enzyme precursors, playing an active role.It is the most specific protease and becomes an indispensable tool in determining the amino acid arrangement of proteins.
Trypsin is a lyophilized preparation that is extracted from the pancreas of cattle, pigs and sheep, purified and crystallized.Easily soluble in water, insoluble in organic solvents such as chloroform, ethanol, ether, etc.At pH1.8, it is almost not inactivated after boiling for a short time;When heated in alkaline solution, it will denature and precipitate, Ca2+The isoelectric point of trypsin is pH10.1.
Bovine trypsin is originally composed of 229 amino acids and contains 6 pairs of disulfide bonds. Its amino acid sequence and crystal structure have been clarified.Under the autocatalysis of intestinal kinase activity, the peptide bond between the N-terminal lysine and isoleucine residue of the zymogen is hydrolyzed, releasing the valium day day day day day lysine 6 peptide, and generating active trypsin.There are 223 amino acid residues of bovine trypsin, with a molecular weight of 23800. The serine residue in the active site is an indispensable serine protease.
Trypsin
The chemical structure of porcine trypsin is very similar to that of bovine trypsin. In the sequence of amino acid residues, only 41 amino acid residues are different, but their molecular configurations are very different.The sedimentation coefficient S20W is 2.77S, pI=10.8, the thermal stability is more stable than that of bovine trypsin, Ca2+The protective effect on enzyme is not as obvious as that of cattle and sheep, without chelating Ca2+The center of the.There are 6 pairs of disulfide bonds and 1~2 bonds are broken, which will not destroy the complete structure of the enzyme molecule and protect the activity of the enzyme.The enzyme activity was equivalent to 72% in cattle and 61% in sheep.
Sheep trypsin is similar to that of cattle and pigs, but its activity is slightly higher than that of cattle and pigs.
Trypsin specifically acts on peptide bond composed of basic amino acid arginine and lysine carboxyl.The enzyme itself is easy to self dissolveβ-Trypsin is converted intoα-Trypsin is further degraded into tryptase and even fragments, and its activity is gradually reduced and lost.
Activation process of trypsinogen
In addition to vertebrates, it also exists in a wide range of organisms such as silkworms, sea pans, crayfish, actinomycetes, etc.In addition, thrombin, plasmin, vasodilator and other proteases related to blood coagulation and inflammation of higher animals are closely related to trypsin in terms of chemical structure and specificity. It can be considered that these enzymes are derived from the evolution of common ancestor enzymes.Chymotrypsin and elastase are also closely related in structure and catalytic mechanism, but their specificity is completely different.
Trypsin degradation analysis
Trypsin is a proteolytic enzyme extracted from the pancreas of cattle, sheep or pigs.According to the Chinese drug standard, the potency per 1mg of dried product shall not be less than 2500 units.An endopeptidase extracted from the pancreas of cattle, sheep and pigs, which only breaks the peptide bond formed by the carboxyl group of lysine or arginine.White or beige crystalline powder.Soluble in water, insoluble in ethanol, glycerin, chloroform and ether.The molecular weight is 24000, pI is 10.5, and the optimal pH value is about 7.8~8.5.PH>9.0 Irreversible inactivation.Ca2+It can stabilize the enzyme activity;Heavy metal ions, organophosphorus compounds, DFP, and natural trypsin inhibitors strongly inhibit its activity.It is clinically used for anti-inflammatory and detumescence, and industrially used in leather manufacturing, raw silk processing, food processing, etc.
Pharmacopoeia information
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This strain is a proteolytic enzyme extracted from pig, sheep or cow pancreas. Calculated as dry product, the activity of trypsin per 1mg shall not be less than 2500 units.
Preparation requirements
This product should be extracted from the pancreas of pigs, sheep or cattle that have passed the quarantine inspection. The species of animals used should be clear. The production process should meet the requirements of the current version of the "Good Manufacturing Practice for Drugs". This product is from an animal source. Virus safety control should be carried out in the process.
character
This product is white or almost white crystalline powder.
identify
Take about 2mg of this product, place it on a white drip plate, add 0.2mL of p-toluenesulfonyl-L-arginine methyl ester hydrochloride test solution, mix it well, and it turns purple.
inspect
acidity
Take this product, add water to dissolve it and make a solution containing 2mg per 1mL. Determine according to the law (general rule 0631), and the pH value should be 5.0~7.0.
Clarity of solution
Take this product, add 0.9% sodium chloride solution to dissolve it and make a solution containing 10mg per 1mL. Check according to the law (general rule 0902 method 1), and the solution should be clear.
Chymotrypsin
It is determined by ultraviolet visible spectrophotometry (general rule 0401).
Substrate solution: TakeN-23.7mg acetyl-L-tyrosine ethyl ester, put into a 100mL volumetric flask, add 50mL phosphate buffer solution (take 38.9mL of 0.067mol/L potassium dihydrogen phosphate solution and 61.1mL of 0.067mol/L disodium hydrogen phosphate solution, mix, pH value is 7.0), heat it to dissolve, cool it, dilute it to the scale, shake it up, freeze it, but do not freeze it repeatedly.
Test solution: Take an appropriate amount of this product, weigh it accurately, add 0.001mol/L hydrochloric acid solution to dissolve it and dilute it quantitatively to make a solution containing 0.25mg per 1mL.
Determination method: Take 2.0mL of substrate solution, 0.2mL of 0.001mol/L hydrochloric acid solution and 1mL of the above phosphate buffer (pH7.0), mix them well, and use them as blank.Precisely measure 0.2mL of the test solution, add 3.0mL of the substrate solution (preheated to 25 ℃± 0.5 ℃), immediately count and shake up, keep the temperature in the colorimetric cell at 25 ℃± 0.5 ℃, read the absorbance every 30 seconds at the wavelength of 237nm for 5 minutes, and the absorbance change rate should be constant every 30 seconds, and the constant time should not be less than 3 minutes, with the absorbance as the ordinate,The time is the abscissa, and the absorbance of the linear part within 3 minutes is taken for drawing, and is calculated according to the following formula.
Where,PIs the amount of chymotrypsin per 2500 trypsin units, unit:;
AtwoIs the absorbance from the straight line;
AoneIs the absorbance terminated on the straight line;
TbyAtwotoAoneReading time, minutes;
WIs the amount of test article contained in the determination solution, mg;
0.0075 means that under the above conditions, the absorbance changes by 0.0075 per minute, which is equivalent to 1 chymotrypsin unit.
Limit: no more than 50 chymotrypsin per 2500 units of trypsin.
Loss on drying
Take an appropriate amount of this product, use phosphorus pentoxide as the desiccant, and dry it under reduced pressure at 60 ℃ for 4 hours. The weight loss shall not exceed 5.0% (General Rule 0831).
Titer determination
It is determined by ultraviolet visible spectrophotometry (general rule 0401).
substrate solution
It shall be used within 2 hours after being madeN-85.7mg of benzoyl-L-arginine ethyl ester hydrochloride, dissolved in water to 100mL, used as the substrate stock solution, 10mL of which was mixed with phosphate buffer solution (take 13mL of 0.067mol/L potassium dihydrogen phosphate solution and 87mL of 0.067mol/L sodium dihydrogen phosphate solution,The pH value is 7.6), dilute to 100mL, keep the constant temperature at 25.0 ℃ ± 0.5 ℃, use water as a blank, and measure the absorbance at the wavelength of 253nm. If necessary, adjust the above substrate stock solution or phosphate buffer solution to make the absorbance between 0.575 and 0.585.
Test solution
Take an appropriate amount of this product, precisely weigh it, add 0.001mol/L hydrochloric acid solution to dissolve it and dilute it quantitatively to make a solution containing 50-60 trypsin units per 1mL.
Assay
Take 3.0mL of substrate solution, add 200 µ L of 0.001mol/L hydrochloric acid solution, mix well, and use as blank.In addition, accurately measure 200 µ L of the test solution, add 3.0 mL of the substrate solution (constant temperature at 25.0 ℃± 0.5 ℃), time it immediately, mix it evenly, keep the temperature in the colorimetric cell at 25.0 ℃± 0.5 ℃ at the wavelength of 253nm, read the absorbance every 30 seconds, a total of 5 minutes.Drawing with absorbance as ordinate and time as abscissa;The absorbance change shall be constant between 0.015 and 0.018 every 30 seconds, and the time of linear relationship shall not be less than 3 minutes.If the above requirements are not met, the concentration of the test solution shall be adjusted and then measured.In the above graph of absorbance versus time, take the absorbance of the linear part and calculate it according to the following formula.
Where,PIs the amount of trypsin contained in each 1mg of test article, unit:;
AoneIs the absorbance terminated on the straight line;
AtwoIs the absorbance from the straight line;
TbyAonetoAtwoReading time, minutes;
WIs the amount of test article contained in the determination solution, mg;
0.003 means that under the above conditions, the absorbance changes by 0.003 per minute, which is equivalent to 1 trypsin unit.
category
Proteolytic enzymes.
Storage
Keep it in a cool and dry place.
preparation
Trypsin for injection.[1]
Brief Introduction to Drugs
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pharmacological action
Trypsin is an endopeptidase that selectively hydrolyzes arginine and lysine peptide chains, and can hydrolyze natural protein, denatured protein, fibrin and mucin into polypeptides or amino acids.Because the serum contains non-specific aprotinin, trypsin will not digest normal tissues, but can decompose mucus secretions such as mucus and purulent sputum, which can promote the penetration of antibiotics and chemotherapy drugs into the focus.
indication
1. It is used for local edema, hematoma and abscess caused by empyema, hemothorax, surgical inflammation, ulcer, traumatic injury, Lou tube, etc.
2. It is used to dissolve mucus and purulent sputum in respiratory diseases.
3. It is used to treat venomous snake bites. More than 800 patients with various types of venomous snake bites, such as Zhuyeqing, Bungarus multicinctus, Cobra, Agkistrodon halys, have been cured.
contraindication
1. Patients with hepatorenal hemorrhage, bleeding tendency and tuberculous abscess should not be used.
2. It should not be used in acute inflammation and bleeding cavity.
Usage and dosage
1. 2.5~5mg each time, dissolve with 5~10mL distilled water.
2. General application: 5000 units once, intramuscular injection once a day, the dosage depends on the circumstances.To prevent pain, appropriate amount of procaine can be added.The local application depends on the situation. It can be prepared into solution preparation (PH7.4 ～ 8.2, the most active when slightly alkaline), spray, powder, ointment, etc., which can be used for intracavitary injection, injection at the affected part, spray, wet compress, and application.
3. Treatment of snake venom: take 1~3 tubes of 2000 units of crystalline trypsin for injection, add 0.25%~0.5% procaine hydrochloride (or water for injection) 4~20mL for dilution, take the tooth scar as the center, and make infiltration injection around the wound, or make circular sealing above the swelling part for 1~2 times.It can be reused if necessary.If the swelling of the injured limb is obvious, the wound can be opened for detoxification and decompression 30 minutes after the injection of this product (except for severe bleeding), or the swelling part can be punctured for detoxification.If the wound is necrotic and festering, apply 0.1% solution to the affected part
matters needing attention
1. No static injection is allowed.Scratch test shall be conducted before use, and it shall be noted that allergic reaction may occur.
2. After absorbing the injection, the needle should be changed to avoid pain during injection.
3. For external use, the injection preparation can be dissolved in buffer solution, but it must be used within 3 hours.
Adverse reactions
1. Local pain and induration after injection.
2. This product can cause histamine release and systemic reaction, including chills, fever, headache, dizziness, chest pain, abdominal pain, rash, angioneurotic edema, dyspnea, elevated intraocular pressure, leukopenia, etc.If the symptoms are mild, the continuous treatment will not be affected. Antihistamines and symptomatic drugs can be given to control the symptoms. If the symptoms are serious, the drug should be stopped immediately.
3. This product can occasionally cause anaphylactic shock.
