Methods and steps for the determination of trypsin activity.

related topics . Analysis of protease activity determination Focus protease research new progress In animal pancreas, trypsin is present in an inactive enzymatic state. Under physiological conditions, trypsin is secreted with pancreatic fluid to the hexa finger intestine, in the small intestine cavity has Ca2 environment, for the intestine
kinase
or trypsin activated, its peptide chain N-end lysine and A peptide bond between isolycine is hydrolyzed, losing an acidic 6 peptide, and its
molecule
configuration changes to a trypsin with catalytic
protein
hydrolysis activity.the molecular weight of
molecule
is about 24,000, its isoelectrine is pH8.9, and the molecular weight of trypsin is about 23400, and its isoelectrine is pH10.8.is most stable at pH3.0, and its thick solution can be stored in the refrigerator (below 0 degrees C) for several weeks without significant loss of activity. pH < 3, trypsin is volatile. Ph> 5, trypsin is self-dissolving. The most appropriate pH for the catalytic activity of trypsin is 7.6 to 7.8.metal ions, organophosphate compounds and reaction products can inhibit the activity of trypsin. Pancreas, egg whites and soybeans also contain proteins that inhibit
pancreatic
activity.trypsin catalyzes the
hydrolysis of proteins and is highly specific to peptide bonds formed by the amino acids
e.g. arginine, lysine) and other amino acids. In addition, trypsin can also catalyse the amamine bonds and ester bonds formed by the base of alkaline amino acids, a high degree of specificity is still reflected in the selection of the alkaline amino acid niche side of the catalytic hydrolysis activity sensitivity of such chemical bonds as: ester bonds >amide bonds> peptide bonds. Therefore, the specific catalytic activity of trypsin can be studied by using a substrate containing any of these chemical bonds.this experimental method uses N-benzoyl-L-arginine ethyl as a substrate, and the hydrolytic reaction is as follows: N-Benzyl-L-Arginine ethyl (BAEE) at wavelength 253nm ultraviolet light absorption is much weaker than N-Benzyl-L-Arginine ultraviolet absorption. Under the catalysis of trypsin, BAEE gradually increases the hydrolysis product BA with the hydrolysis of the ester bond, and the ultraviolet light absorption of the reaction system increases accordingly, and the activity of trypsin is calculated by A253nm.BAEE unit of trypsin is defined as: BAEE as the substrate, under certain reaction conditions, the amount of enzyme per minute to increase A253nm by 0.001 is one BAEE unit.method ii uses casein as the base method to determine the activity of trypsin. Casein as the base, mainly used to determine the vitality of trypsin crude products. Tryptoprotein catalytic hydrolytic substrate casein produces
small molecules
peptides and amino acids that are not precipitated by triclosan acetic acid. Within a certain concentration range, the value added of the enzyme's hydrolytic filter at wavelength 275nm is directly related to the vitality unit of trypsin. The light absorption of the standard tyrosine solution was used as a control, and the activity of trypsin in the sample was determined according to the definition of the unit of vitality. Definition of the unit of vitality: Under certain conditions, the value-added of light absorption per minute of the enzyme hydrolytic filter is one protease unit (U) when the value of light absorption is the same as that of 1 sm;mol tyrosine at 275nm.1,
Reagents(1) 1.0mmol/LBAEE substrate solution(2) 10mmol/LHCl(3) 0.1mol/L, pH8.0 borate buffer(4) 10mg /mL casein solutiontake casein 1g, add 13mL0.1mol/LNaOH, distilled water 40mL, put 60 degrees C water bath
heat
to dissolve, put to room temperature, dilute the water to 8.0 and set the capacity to 100mL.(5) 5% triclosan solution(6) 0.2mol/L hydrochloric acid solution(7)50-mL casein control solution accurately weighed by 105 degrees C
dry
to constant tyrosine 5mg, with 0.2mol/L hydrochloric acid to 100l. 2, equipment (1)
ringing temperature
water bath (2) ultraviolet
hydrodometer (3)
centrifuge (4) test tube (methods and procedures) 1, N-benzoyl-L-arginate ethyl (BAEE) assay: takes 2 covered quartz color cups with a light range of 1cm, and adds a warm-up 2.8mL 1.0mmol/L BAEE substrate solution at 25 degrees C. Add 0.2mL10mmol/L HCl to one color cup, adjust the instrument zero point at wavelength 253nm as a blank, add 0.2mL enzyme fluid to another color cup, immediately cover with a quick mixing time, read every half minute, read a total of 3 to 4min. The measured results should be suitable for the control of A253nm/min between 0.05 and 0.100. If you deviate from this range, increase or decrease the amount of enzymes appropriately. with time (t) as the horizontal coordinates, the light absorption value (A253nm) as the ordinate for the graph, in the straight line part of any time interval and the corresponding light absorption value change (A253nm), according to the following formula to calculate the vitality unit of trypsin. 2, casein for substrate determination (1) take 3 test tubes, 1 to 3 numbers. test tube 1, add 1.0mL enzyme solution, add 0.1mol/L, pH8.0 borate buffer 2.0mL, in the water bath insulation 10min, precisely add 40 degrees C preheated 10mg/mL casein solution 5.0mL, mixed well, immediately placed in a 40c water bath, accurate reaction 30min, add 5.0mL of 5% triclosan acetic acid, quickly shake well. test tube 2, add 1.0mL enzyme solution, add 0.1mol/L, pH8.0 borate buffer 2.0mL, in 40 degrees C water bath insulation 10 min, precisely add 5.0mL 5% triclosan acetic acid, shake well, in 40 degrees C water bath insulation 30min, then add 10mg/mL casein solution 5.0mL, mix well. test tube 3 and mixed with 5.0mL 5% tricloste and 8.0mL 0.1mol/L (pH8.0) boric acid buffer, respectively. the above three test tube solution at 3,500r/min centrifugation 10min, respectively, to take the liquid. the light absorption values of test tube 1 and test tube 2 centrifugal liquid (A275) were measured at wavelengths of 275nm in the UV hydrodometer, using test tube 3 centrifugal liquid as a parameter. (1) determination of tyrosine control solution with 0.2mol/L hydrochloric acid solution as a reference, at a wavelength of 275nm to determine the light absorption value (AS) of tyrosine control solution. the light absorption value A-Test Tube 1 in the formula of the test tube. . A0 - the light absorption value measured by test tube 2; . AS - the light absorption value of tyrosine control solution; . C-control - concentration of tyrosine ( sum;g/mL); . M-control - molecular weight of tyrosine (181.19); . V total - total reaction volume (mL); . V sample - added sample liquid product (mL); t - reaction time (min); n - a dilution multiply of the sample.
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