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Experiment 9.

Purpose

Activity Assay of Alkaline Protease

1. To grasp the principle of alkaline protease activity assay and the computation method of protease activity. 2. To learn the approach and the basic operation of enzyme-catalyzed reaction speed assay.

Principle
Enzyme activity is the capability of catalyzing some chemical reaction. The value of enzyme activity may be expressed by the speed of some chemical reaction that catalyzes under certain conditions. Enzyme activity assay is to assay the speed of chemical reaction catalyzed by enzyme in reality. The speed of enzyme-catalyzed reaction may be expressed with the decrease of reaction substrate or the increase of product in unit time. Usually, assaying the resulting volume of the derivatives within per unit time is applied for the sake of sensitivity. Since enzyme-catalyzed reaction speed may be reduced along with time, we must assay the initial speed of enzyme-catalyzed reaction so as to assay enzyme activity correctly. Alkaline protease may catalyze casein to hydrolyze and generate tyrosine under the alkaline conditions. Tyrosine is aminophenol which contains phenolhydroxyl and may cause Folin-phenol reaction with Folin reagent (a mixture of phosphowolframate and phosphomolybdate). (Folin-phenol reaction: Folin reagent is very unstable under alkaline conditions; it is apt to be deoxidized by phenol combinations quantificationally and creates a mixture of wolfram blue and molybdenum blue that wears various shades of blue.) By way of colorimetry, the formation volume of tyrosine can be assayed. Express enzyme activity with the amount of tyrosine derived from hydrolyzed casein.

Materials
1. Apparatus Electric-thermostatic water bath trough, Assay scale, Volumetric flask, Tubes for solution transfer, Tubes and tube shelf, Glass funnels, 721 spectrophotometer 2. Reagent (1) Folin reagent: Add 50g of Na2 WO4 2H2 O, 125g of Na2 MoO4 2H2O, 350ml of distilled water, 25ml of 85% H3 PO4 and 50ml of concerntrated HC1 into a 1L round- bottom stoppered flask, mix well and reflux l0h. After refluxing, add 25g of Li2 SO4 , 25ml of distilled water, several drops liquid Br2 , boil 15min without cap to get rid of excessive Br2 . After cooling, dilute to a constant volume of 500ml by volumetric flask, filtrate, keep the filtrate in brown flask in darkroom. Add 4 times diluted water before using. (2) 1% casein solution: Weigh 1g casein in a mortar. Make it humid with a dollop of distilled water. Then add slowly 0.2 mol/L NaOH 4ml and grind it adequately. Wash it out into a volumetric flask. Put it into water tub and boil it 15 minutes. When it is cooled off, set the volume at 100ml and then store it into a refrigerator. (3) pH10 buffering solution: Solution A (0.05 mol/L borax solution): Weigh 19g borax (Na2 B4O 7 2 O). Dissolve it in 10H distilled water and set the volume at 1000ml. Solution B :0.2 mol/L sodium hydroxide solution Decoct pH10 borax sodium hydroxide solution: Sip 50ml of Solution A Add in 21ml Solution

B. Set the volume at 200ml with distilled water. (4)Standard tyrosine solution: Weigh 50mg tyrosine precisely and add 1ml 1mol/L hydrochloric acid. When it is dissolved, set the volume at 50ml. Then we have 1mg/ml standard solution of tyrosine. (5) 0.4mol/L sodium carbonate solution, 0.4mol/L trichloroacetic acid solution.

Procedures
1. Standard curve of tyrosine preparation (1) Prepare 7 tubes, number them, and then decoct tyrosine solution of various contents: Tube No. 0 1 2 3 4 5 6 Content of tyrosine g 0 100 200 300 400 500 600 1mg/ml standard tyrosine solutionml 0 0.1 0.2 0.3 0.4 0.5 0.6 Distilled water (ml) 2.0 1.9 1.8 1.7 1.6 1.5 1.4

(2) Add 1% 1 ml casein solution into the above-mentioned 7 tubes. Keep it in water bath at 40 for 15 minutes to preserve heat. After taking it out, add 0.4mol/L trichloroacetic acid 3ml, C and shake it well. Each tube should be filtered with filter paper. (3) Sip 1ml filtered solution for each of the 7 tubes, respectively. Add 5ml 0.4mol/L sodium carbonate solution, 1ml Folin reagent, and shake well. Keep it in water bath at 40 for 15 C minutes to preserve heat. Then add 3ml distilled water in each of the tubes and shake it well. (4)Assay the optical density at 680nm with 721 spectrophotometer as contrasted with Tube 0. (5) Draw standard curve by taking optical density as vertical ordinate and the tyrosine content (in microgram) as abscissa. 2. Sample assay (1)Weigh 2 grams of dry enzyme powder, add in 10ml pH10 buffering solution. Let it dissolve in a small beaker, stir it with a glass rod. After moments of its slack, dump the upper layer of solution into the volumetric flask carefully. Add a touch of buffering solution to the residue. Stir the solution four times as such. Remove all of it into the 200ml a volumetric flask at last. Set the solution to the mark with buffering solution, shake it well, filter it with 2-layer or 4-layer gauze, imbibe 5ml filtrate solution, and transfer it into 100ml volumetric flask. Dilute it with distilled solution and then what we have is enzyme solution that has been diluted 2000 times. (2)Take out 3 dry tubes. Number them in line with the following table. Add reagent and operate in strict accordance with the order in the table. Shaking it well, filter each tube respectively. Imbibe 1ml solution, add 5ml 0.4mol/L sodium carbonate solution, 1ml Folin reagent, shake it up forcefully. Keep it in the water bath at 40 for C 15 minutes to preserve heat. Afterwards, add 3ml distilled water into each tube, shake it well. Assay the optical density of the two tubes at wavelength 680nm with 721 spectrophotometer in antithesis to the contrasted tube.

Tube No. Reagent PH10 Buffering solution (ml) 1:2000 Alkaline protease (ml) 0.4mol/L Trichloroacetic acid solution (ml) 1% Casein solution (ml) 40 Water bath heat preservation (min) 0.4mol/L Trichloroaceticacid solution (ml)

Contrast 1 1 3 1 15 0

1 1 1 0 1 15 3

2 1 1 0 1 15 3

Results
1. The definition of the activity unit of alkaline protease of this experiment: 1g alkaline protease under the conditions of pH10, 40 and hydrolyzed casein may produce C, 1mg tyrosine each minute that is defined to be 1 enzyme activity unit. 2. The computation of the activity unit of alkaline protease of this experiment: Activity unit of each gram of alkaline protease = m / t f m: the value of the optical density assayed from the sample. Check the standard curve and we have the volume of tyrosine ( g). t: time for enzyme-catalyzed reaction f: the dilution multiple of enzyme. In this experiment, f = 2000

Questions
1. What is the activity of enzymes? How to computate the activity of enzymes? 2. What are the attentions in the alkaline protease activity assay?