Method of Enzyme Assay. Arwa AL-Khyyat. KSU. Biochemistry

Transcription

1 Method of Enzyme Assay

2 Method of Enzyme Assay 1- End point assay: Alanine transaminase (colorimetric\ endpoint assay) 2- Continuous assay: Glucose-6-phosphate dehydrogenase ( UV/ kinetics)

3 Objective To study the different methods for determining enzyme activity.

4 Enzyme assays are laboratory methods for measuring enzymatic activity. All enzyme assays measure either the consumption of substrate or production of product over time. S E P + E

5 Spectrophotometric methods Fluorescence methods Sampling methods Manometric methods Eletrode Methods Polarimetric Method

6 Enzyme assays can be split into two groups Continuous assays, where the assay gives a continuous reading of activity. Discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.

7 Spectrophotometer: In spectrophotometric assays, you follow the course of the reaction by measuring a change in how much light the assay solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, these are called colorimetric assays

8 Absorbance measurement for ultraviolet or visible light. Absorbance is measured in a spectrophotometer, which provides a light source with selectable wavelength in the range nm (ultraviolet and visible light). Sample is contained in a cuvett of exactly 1.00 cm thickness. The light passing through the sample is then measured and recorded by a detector.

9 spectrophotometer

10 1- cases in which product absorb but not the substrate. e.g. Fumarate malate 2- the Co-enzyme undergose change in absorption on reduction or oxidation by S Oxidized form NAD NADP FAD Reduced form NADH NADPH FADH2

11 End point assay Alanine transaminase (colorimetric\ endpoint assay) Transaminase or an aminotransferase is an enzyme that catalyzes a type of reaction between an amino acid and an α- keto acid. Specifically, this reaction (transamination) involves removing the amino group from the amino acid, leaving behind an α- keto acid, and transferring it to the reactant α-keto acid and converting it into an amino acid.

12 The enzymes are important in the production of various amino acids, and measuring the concentration of various transaminases in the blood is important in the diagnosing and tracking many diseases. Alanine transaminase or ALT is a transaminase enzyme. It is also called: Serum glutamic pyruvic transaminase (SGPT) or Alanine aminotransferase (ALAT(.

13 ALT is found in serum and in various bodily tissues, but is most commonly associated with the liver. Function : It catalyzes the transfer of an amino group from alanine to a-ketoglutarate, to form pyruvate and glutamate under controlled condition (37 C) and ph 7.4 ± Alanine + a-ketoglutarate = pyruvate + glutamate

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15 Use of Co-enzyme in Assay UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in their reduced forms, but do not in their oxidized forms. An oxidoreductase using NADH as a substrate could therefore be assayed by following the decrease in UV absorbance at a wavelength of 340 nm as it consumes the coenzyme. NAD+ does not absorb Ultraviolet at 340 nm. NADH strongly absorbs Ultraviolet at 340 nm. Rate can be measured as disappearance of reactant or accumulation of product. If NADH product: increase the absorbance / min If NADH substrate: decrease the absorbance / min

16 NAD+ does not absorb Ultraviolet at 340 nm NADH strongly absorbs ultraviolet at 340 nm.

17 Continuous assay: Glucose-6-phosphate dehydrogenase ( UV/ kinetics) The enzyme G6PD (old name G6P-DH) catalyses the dehydrogenation of glucose -6-phosphate as the first step in pentose phosphate pathway. NADP+ the electron acceptor, is reduced to NADPH in the reaction. The ph optimum for the enzyme reaction is 8.3 for the enzyme from yeast or blood cell.

18 Principle of method (colorimetric endpoint procedure): In this method ALT catalyzes the reaction of L-alanine and - ketoglutaric acid to form pyruvate and glutamate under controlled conditions (37 C) and ph Alanine + a-ketoglutarate = pyruvate + glutamate The addition of acidic 2,4-dinitrophenylhydrazine stops the reaction and forms the 2,4-dinitrophenylhydrazone. So that it may be measured at nm. (ALT is assay by following formation of pyruvate from alanine using 2, 4-dinitrophenylhydazine at alkaline ph)

19 Material and Method End point assay Alanine transaminase (colorimetric\ endpoint assay) Material ALT SUBSTRATE: 0.2 M L-Alanine, 2.0 mm -Ketoglutaric acid, 100 mm Phosphate buffer at ph Also contains 0.2 % v/v Preservative. Keep tightly capped and protected from contamination. Store at 2-8 C. COLOR REAGENT: 1.0 mm 2,4-Dinitrophenylhydrazine in 1 N hydrochloric acid Also contains 0.2% v/v preservative. Keep tightly capped and protected from excessive exposure to direct sunlight. Store at 2-8 C. 3. COLOR DEVELOPER: 0.5 N Sodium hydroxide. Keep tightly capped and protected from excessive exposure to atmospheric CO 2 Can be stored at room temperature.

20 PRECAUTIONS COLOR REAGENT contains 1 N Hydrochloric acid which causes burns. In case of contact, flush affected area with large amounts of water. Seek medical attention. COLOR DEVELOPER contains 0.5 N Sodium hydroxide which is corrosive. In case of contact, flush affected area with large amounts of water. Seek medical attention.

21 PROCEDURE: BLANK SAMPLE ALT Substrate 0.5 ml 0.5 ml Pre-warm at 37 C for 5 minutes and add: (using timed intervals) Distilled Water Sample 0.1 ml ml Mix, and incubate at 37 C for exactly 30 minutes (use same timed intervals), and add: Color Reagent 0.5 ml 0.5 ml Mix, and return at 37 C for exactly 10 minutes, then add: (use same timed intervals) Color Developer 5.0 ml 5.0 ml Mix, and return to 37 C for exactly 5 minutes. Read absorbance of all tubes at 546nm against blank.

22 STABILITY OF ENDPOINT REACTION The Final color produced in the reaction should be measured within 60 minutes. Results: The data shown in the table below is used to convert absorbance at 546 nm into enzymatic activity in U/L of serum. Draw graph using the data in table with absorbance on the Y- axis and enzymatic activity in U/L on the X-axis. Record your result in the spaces provided below the table. Absorbance at 546 nm ALT (SGPT) activity Absorbance at 546 nm = ALT (SGPT) activity of serum sample (from graph)= U/L

23 Continuous assay: Glucose-6-phosphate dehydrogenase ( UV/ kinetics) PRINCIPLE: The rate of formation of NADPH is a measure of the G6P- DH activity and it can be followed by means of the increase in extinction at 340. The overall reaction is outlined as follows: Glucose-6-phosphate + NADP + G6P-DH 6Phosphogluconate + NADPH + H +

24 Continuous assay: Glucose-6-phosphate dehydrogenase ( UV/ kinetics) Material REAGENTS: G6P-DH BUFFER: Triethanolamine buffer 50 mmol/l, EDTA 5 mmol/l, ph (25 o C). Preservative added. NADP REAGENT: 30 mm NADP. G6P-DH SUBSTRATE: Glucose-6-Phosphate Sodium Salt 17 mm.

25 Procedure (manual) Note: Pipette into clean and dry test tubes: FOR SERUM/PLASMA G6P-DH Buffer 3.0 ml NADP Reagent 100 l Hemolysate RBC (supernatent) 50 l Mix and incubate for 5 minutes at 25 o C, then add: G6P-DH Substrate 50 l Mix and transfer to cuvette at 25 o C. After 30 seconds read initial absorbance at 340 nm against distilled water(blank). Repeat absorbance readings every minute for the next 3 minutes and calculate the mean A/min.

26 Results TUBE A B C D TIME 30 SECONDS 1.5 MIN 2.5MIN 3.5 MIN ADSORBANCE 340nm CALCULATIONS A1 = (C-B) /1 A2=(D-C) /1 ( A2+ A1)/2 = A A/min x = G6P-DH activity in Um/ erthrocyte per ml of blood

27 Glucose-6-phosphate dehydrogenase (G6PD) is a critical enzyme for preventing free radical damage in red blood cells. An estimated 400 million people worldwide, however, are deficient in this enzyme due to a genetic mutation. Under certain circumstances G6PD deficiency can lead to destruction of red blood cells and health consequences that vary from mild to severe. G6PD deficiency occurs most frequently in certain parts of Africa, Asia and the Mediterranean. More than 100 mutations can cause G6PD deficiency. The form of G6PD deficiency caused by this mutation is only rarely associated with sensitivity to fava beans.