Brief Methodologies For Analysis Of Different Enzymes

Brief Methodologies for Analysis of Different Enzymes Dr. Subhendu Datta Sr. Scientist Kolkata, India ASPARTATE AMINO TRANSFERASE (Wootton, 1964 as described in Practical Biochemistry by Dr. Murtuza. First Edition: 1998) The substrate consisted of 0.2M D,L – aspartic acid and 0.002M solution of alpha keto glutarate in 0.05 M Phosphate buffer (pH7.4). The experimental and control tubes 0.5 ml of substrate was added and the reaction was started by addition of 0.1 ml of liver homogenate (1 in 50 dilutions). The mixture was incubated (370C) for 60 minutes and the reaction was stopped by addition of 0.5 ml of 0.001 M 2,4-dinitrophenyl hydrazine (DNPH).In the control tube the enzyme source was added after the addition of DNPH solution. The tubes were kept at room temperature for 20 minutes with occasional shaking. Then 5 ml of 0.4N NaOH was added and mixed thoroughly. After 10 minutes OD was recorded at 540nm. ALANINE AMINO TRANSFERASE (Wootton, 1964 as described in Practical Biochemistry by Dr. Murtuza. First Edition: 1998) The procedure adopted was the same as described for aspartate amino transferase except that substrate comprised 0.2 M DL alanine instead of aspartic acid. LACTATE DEHYDROGENASE (Wrobleuuiski and Ladue, 1955 as described in Practical Biochemistry by Dr. Murtuza. First Edition: 1998)

The LDH activity was assayed by the method of Wroblewski and LaDue (1955). The total 3 ml reaction mixture comprised 2.7 ml of 0.1 M phosphate buffer (pH 7.5), 0.1ml of NADH solution (2 mg NADH dissolved in 1ml of phosphste buffer sol.), 0.1 ml of the tissue homogenate, and 0.1 ml of 0.2 M sodium pyruvate. The was started after the addition of substrate (sodium pyruvate). The OD was recorded at 340 nm, every 30 seconds. The enzymic activity was expressed as units/mg protein/min, at 250C, where 1 unit was equal to ∆ 0.01 OD/min. MALATE DEHYDROGENASE (Ochoa, 1955 as described in Practical Biochemistry by Dr. Murtuza. First Edition: 1998) The MDH activity was assayed by the method of Ochoa (1955). The total 3 ml reaction mixture comprised 2.7 ml 0.1 M phosphate buffer (pH 7.5), 0.1 ml of concentrated 1

NADH sol. (2.0 mg NADH dissolved in 1ml of phosphste buffer sol.), 0.1 ml of the tissue homogenate, and 0.1 ml of oxaloacetate sol. (2 mg oxaloacetate dissolved in 2ml of chilled G.D.W). The OD was recorded at 340 nm at 30 seconds intervals, for 3minutes. Distilled water was used as the blank. The MDH activity was expressed as units/mg protein/min, at 250C, where 1 unit was equal to ∆ 0.01 OD/min. ACETYLCHOLIN ESTARASE (Augustinsson, 1957 as described in Practical Biochemistry by Dr. Murtuza. First Edition: 1998) The enzyme was assyed by the method of Hestrin (1949), modified by Augustinsson (1957). The AchE assay system (2.1ml) comprises 1.0 ml of 4mM acetyl Choline (pH 4.0) substrate buffer mixture (1:9) and 1.0 ml of M/15 phosphate buffer (Ph-7.2). Before adding the substrate 1.0 ml of the buffer solution was taken and 0.1 ml of the homogenate (10%) was added. The contents of the tubes were equilibrated (37oC) for two minutes. The substrate (acetylcholine) was added and incubated (37oC, 30 minutes). Adding 2.0 ml of the alkaline hydroxylamine solution terminated the reaction and the contents were mixed well. The enzyme source was then added to the control tubes. Colour was developed by adding 1.0 ml of 10% ferric chloride solution and 1ml of 1:2 HCl solution to each contents were filtered and the OD was recorded at 540 nm against the reagent blank set to zero density. QUANTIFICATION OF PROTEIN (Lowry, et. al., 1951 as described in Practical Biochemistry by Dr. Murtuza. First Edition: 1998) The protein estimation was assayed by the method of Lowry, 1951. The assay system consisted of two steps, Preparation of sample and Estimation of protein. In the preparation ml sample was taken then added 1 ml 10% T.C.A. Then the contents were centrifuged (5000 rpm for 20 minutes). The supernatant was discarded and dissolved the residue with addition of 0.5 ml 0.1(N) NaOH. After dissolving 0.1 ml sample was taken for protein estimation and added 5 ml Alkaline CuSO4. 0.5 ml of Folinn reagent (1N) was added after waiting 10 minutes. Then the mixture was incubated at room temperature in dark place for 30 minutes and OD was recorded at 660 nm. In the Blank and standard the procedure was same except 0.1 ml BSA (10mg/50ml) was taken in standard instead 0.1 sample. ALKALINE PHOSPHATASE (Lowry, et. al., 1954 as described in Practical Biochemistry by Dr. Murtuza. First Edition: 1998) The enzyme was assayed by the method of Lowry et. al., 1954. The total volume of the assay system was 2 ml. The experimental tubes contained 0.2 ml bicarbonate buffer, MgCl2 0.1 ml, enzyme 0.1 ml, Distilled water 0.5 ml and 0.1 ml substrate (pnpp). The mixture was incubated 370C for 15 minutes. Adding 1 ml NaOH terminated the reaction.

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In the control tube, the enzyme source was added after addition of NaOH. The OD was recorded at 410 nm. ACID PHOSPHATASE (Lowry, et. al., 1954 as described in Practical Biochemistry by Dr. Murtuza. First Edition: 1998) The procedure adopted was the same as described for alkaline phosphatase except the buffer comprised acetate instead bicarbonate buffer.

REFERENCES Methodology

References

Aspartate Amino Transferase & Alanine Amino Transferase

Wotton (1964). I.D.P. Microanalysis In Medical Biochemistry. 4th edn. J. & A. Churchill, London PP. 101-107.

Lactate Dehydrogenase

Wrobleuiski and Ladue (1955). LDH activity in blood. Proc. SOC. Exp. Biol. Med., 90: 210-213.

Malate Dehydrogenase

Ochoa (1955). Malic dehydrogenase and ‘malic’ enzyme In Methods of enzymology Vol.1 Coloric, S.P. and Kaplan, N. (eds), Academic Press, Newyork, pp 735-745.

Acetyl Choline Esterase

Hestrin 1949 modified by Augustinsson (1957). The reaction of Acetyl choline esters and other Carboxylic acid derivatives with Hydroxylamine and its analytical application. J. Biol. Chem., 180: 249-261.

ALP& ACP

Lowry, et.al. (1954). J. Biol. Chem. 207. 19, as described by S. Sadasivam & A. Manickam in Biochemical methods, 2nd edition.

Quantification of Protein By Lowry method

Lowry, O. H. et. al., (1951). Protein measurement with Folin Phenol Reagent. J. Biol. Chem. 193: 265-276.

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