The preparation and determination of the activity of trypsin.

related topics Analysis of protease activity assays New advances in focused protease researchThe purpose requires (1) to learn the basic methods of purification and crystallization of trypsin.(2) to understand the concept of enzyme activity and ratio activity.Experimental PrincipleTrypsin is present in animal pancreas in the form of inactive enzymes, in the presence of Ca2 plus, activated by the intestine
kinase
or active trypsin itself, from the peptide chain N end lysine and isocyminine residue between the peptide bonds disconnected, the loss of a hexeptide, molecular structure after a certain change into an active trypsin.the molecular weight of tyrinase is about 24,000, its isoelectrine is about pH8.9, the molecular weight of trypsin is close to its enzyme (23300), its isoelectrine is about pH10 .8, the most suitable pH7.6 to 8.0, at pH s3 when the most stable, lower than this pH, trypsin susceptible, in pH>5 when easily self-dissolving. Ca2 plus ions have a stable effect on trypsin.heavy metal ions, organophosphate compounds and reactants can inhibit the activity of trypsin, which is found in the seeds of pancreas, egg whites and legumes. It has recently been found that trypsin inhibitors are also present in the block base of some plants (e.g. potatoes, sweet potatoes, taro, etc.).trypsin catalyzes the hydrolysis of
proteins
and is highly specialized in the bonds formed by the amino acids
(arginine, lysine) of alkaline
amino acids and amino acids. In addition, it can also catalycify the formation of alkaline amino acids and carboxyl alamide bonds or ester bonds, its highly specialized is still reflected in the choice of one end of the alkaline amino acids.
the sensitivity of trypsin to these bonds is: ester bond> and > peptide bond. Therefore, acrylamides or ester compounds containing these bonds can be used as substrates to determine the vitality of trypsin. At present, benzoyl-L-arginine-nitrobenzene (BAPA) and benzoyl-L-arginine-β-amine (BANA) are commonly used to determine the vitality of amylamine. The vitality of esterase was determined by paraben-L-arginine ethyl (BAEE) and paraben sulfonyl-L-arginate (TAME). In this experiment, BAEE was used as the substrate, and the activity of trypsin was determined by ultraviolet absorption. The requirements of enzyme vitality units often vary according to substrates and assay methods.When extracting trypsin from an animal's pancreas, it is usually extracted from the enzymes contained in pancreatic cells with a thin acid solution, and then, according to the principle of isoelectric precipitation, adjusts pH to precipitate and remove a large number of acidic hemroteins and non-protein hems. Quality, and then ammonium sulfate graded salt analysis of trypsinogen (including a large number of acidic impurities and non-protein impurities, and then ammonium sulfate graded salt analysis of trypsinogen and so on (including a large number of protease and elastinase) precipitation.
After dissolution, it is activated with a very small amount of active trypsin, which transforms its enzymatics into active trypsins (the protease and elastinase are also activated), and the activated enzyme solution is then removed by salt analysis grading. The grades containing trypsin were collected and further purified by crystallization. Generally after 2 to 3 crystallization, a fairly pure trypsin can be obtained, which can reach 8000 to 10000 BAEE units / mg protein, or higher than vitality.if a more pure preparation is needed, the above enzyme solution can be purified by affinity layering method.reagents
and equipment 1, reagents pH2.5 acetate water; 2.5mol/L H2SO4; 5 mol/L NaOH; 2 mol/L NaOH; 2mol/L HCl; 0.001M HCl; ammonium sulfate; calcium chloride.0.8 mol/L pH9.0 boric acid buffer: take 20 ml 0.8 mol/L boric acid solution, plus 80 ml 0.2 mol/L sodium tetrate solution, after mixing, check the correction with a pH meter.0.4 mol/L pH9.0 boric acid buffer (1x dilution with 0.8 mol/L);0.2 mol/L pH8.0 boric acid buffer: take 70 ml 0.2 mol/L boric acid solution, plus 30 ml 0.5 mol/L sodium tetrate solution, after mixing, with pH meter correction. 0.05 mol/L pH8.0 Tris-HCl buffer: Take 50mL 0.1 mol/LTris plus 29.2 mL mol/L HCl plus water to 100mL. of the substrate solution: i.e. 0.34 mg BAEE and 2.22 mg calcium chloride per milliliter of 0.05 mol/L pH8.0 Tris-HCl buffer. II, materials fresh or frozen pig pancreas3, instruments food processors and high-speed dispersors; research; large glass funnel; cloth funnel; filter bottle; gauze;
water bath
temperature, ultraviolet hydrometer, stopwatch, pH test paper. Operating Methods First, pig trypsin preparation (i) extraction of pig trypsin original Pig pancreas 1.0Kg (fresh or refrigerated immediately after killing), after removing fat and connective tissue, stranded. 2x volume pre-cooled acetic acidified water (pH2.5) in 10 to 15 degrees C stirring extraction for 24 hours, four layers of yarn b
filtration
milk White filter, with 2.5M H2SO4 pH to 2.5 to 3.0, placed for 3 to 4 hours with folding filter paper filter yellow transparent filter (about 1.5 liters). Add solid ammonium sulfate (to study first), so that the solution up to 0.75 saturation (492 grams per liter of filter) placed after night filtration (squeezing dry), pig trypsin raw crude products. (ii) Trypsin original activation to trypsin original coarse product filter cake sub-add 10 times the volume (re-counting by cake) cold distilled water, so that the filter cake solution dissolved, trypsin original solution. The fine solid aqueous calcium chloride is slowly added to the original solution of the enzyme (the content of ammonium sulfate in the filter cake is by a quarter of the weight of the cake), so that Ca2 plus SO42-binding, side by side stirring well, side by side stirring, so that the solution still contains 0.1M CaCl2. . 2. Use 5M NaOH to adjust pH to 8.0, add a very small amount of pig trypsin (about 2-5mg) to stir gently, at room temperature to active for 8 to 10 hours, (2 to 3 hours sampling once, and diluted with 0.001M HCl), to determine the increase in enzyme activity. . 3. After the reification is completed (about 3500 to 4000 BAEE units), the CaSO4 precipitation is removed by 2.5M H2SO4 to 2.5 to 3.0. (iii) Separation of trypsin 1. The activated trypsin solution is added to the fine powdered solid ammonium sulfate at 242 g/L, so that the solution reaches 0.4 saturation, and after a few hours, the filter is pumped and the filter cake is discarded. . 2. The filter is added to the fine ammonium sulfate at 250 g/L, so that the solution satiety reaches 0.75, and is placed for several hours, the filter is pumped and the filter is discarded. (iv) Crystallization of trypsin 1. After the dissolution of the above-mentioned trypsin filter cake (crude trypsin): the buffer is added to the buffer according to the measure of 0.4M boric acid buffer dissolved in 1.0 ml pH9.0 per cleaf. . 2. With 2M NaOH tune pH to 8.0, pay attention to careful adjustment, partial acid is not easy to crystallize, partial alkali fragile, stored in the refrigerator. 3. After a few hours of placement, a large number of flocs should appear, the solution gradually thickened into a glial state, and then add a total volume of 1/4 to 1/5 pH8.0 of 0.2M boric acid buffer, so that the glial dispersion, if necessary, add a little trypsin crystal. . 4. Placed for 2 to 5 days can get a large number of trypsin crystallization, to be crystallization out completely, filtration, mother fluid recovery. (v) The recrystration of trypsin recrystrystase products for the first crystallization: with about 1 times the 0.025M HCl, the above crystallization is dispersed, adding about 1.0 to 1.5 times the volume of pH9.0 of the 0.8M boric acid buffer, to Crystallase all dissolved, sampled, with 2M NaOH modulation solution pH to 8.0 (accurate) (too large, difficult to crystallize), refrigerator placed for 1 to 2 days, can be a large number of crystallization filtered to the second crystallization product (mother fluid recovery), frozen
drine
after the recrystimulation of pig trypsin. second, the determination of trypsin activity with benzoyl L- arginate ethyl ethyl (BAEE) as the substrate, using ultraviolet absorption method for determination. UV absorption of benzoyl L-arginine at wavelength 253nm is much weaker than benzoyl L-arginine (BA). Under the catalysis of trypsin, with the hydrolysis of ester bonds, benzoyl L-arginine gradually increases, and the UV absorption of the reaction system should increase accordingly. two caped quartz color cups with a light range of 1 cm were taken and a 2.8 ml substrate solution with a heat of 25 degrees C was added. Add 0.2 ml 0.001mol/L HCl to a color cup to correct the light absorption zero point at 253nm of the instrument as a blank. Add another 0.2 ml of the enzyme fluid to be tested (the amount is generally 10 micrograms of crystalline trypsin) to the other color cup, immediately mix and remember, read every half minute, read a total of 3 to 4 minutes. It is appropriate to control the DA253/min at around 0.05 to 0.100. the enzyme-driven reaction dynamics curve, from the curve to find the reaction starting point absorbent rate over time (i.e., the initial velocity) DA253/min. . The definition of trypsin vitality unit is as: BAEE as the substrate reaction fluid pH8.0, 25 degrees C, reaction volume 3.0 ml, optical diameter of 1 cm conditions, the determination of DA253, per minute to increase DA253 0.001, the amount of enzyme added to the reaction fluid is one BAEE unit. Note (1) Pancreas must be freshly slaughtered tissue or stored at low temperature immediately, otherwise the experiment may fail due to self-dissolving tissue. (2) at room temperature 14 to 20 degrees C conditions 8 to 12 hours can be activated completely, activation time is too long, because the enzyme itself self-dissolving will make the ratio of life reduced, than the activity reached "3000 to 4000 BAEE units / mg protein" can stop activation. (3) In order to obtain trypsin crystallization, in the crystallization should be very careful in accordance with the prescribed conditions, do not be careless, the first few steps of separation purification effect is better, then
cedation
crystallization is also easier, so each step of the operation should be strict. Enzyme protein solution is too thin to form crystallization, too thick is easy to form amorphoous precipitation precipitated, therefore, it is necessary to be just right, in general, the solution to be crystallization should be slightly cloudy at the beginning. (4) overacid can affect the formation of crystallization and enzyme vitality changes, it is necessary to strictly control PH. (5) the first crystallization, 3 to 5 days after the still no crystallization, should check pH, if necessary, adjust pH or inoculation, to promote crystallization formation. Recrystrying time is shorter
.