Question

Pick any two enzymes for this question. Answering this effectively may require creativity and insight!

a. Describe how you might separate them from each other using techniques that you have read about, are using in lab, or that you have used elsewhere. (ex. size exclusion chromatography)

b. Indicate how you could determine that you had successfully separated them.

c. Describe the protocol you would use to assay them for activity.

Answer 1

Superoxide dismutase

Principle: SOD (EC 1.15.1.1) dismutates O2. to H2O2 and O2. The present assay is based on an indirect method involving photo reduction of riboflavin which generates O2.. It is allowed to react with hydroxylamine hydrochloride to produce nitrite. The nitrite reacts with sulfanilic acid to produce a diazonium compound that subsequently reacts with naphthylamine forming a red azo compound which absorbs light at 543 nm. When SOD present in the assay mixture, nitrite formation is inversely proportional to the amount of SOD as it scavenges O2. competitively with nitrite. One unit of enzyme activity was defined as amount of SOD capable of inhibiting 50% of nitrite formation under assay conditions.

Reagents: 1) Tris buffer, pH 8 in DW (100 mM). 2) L-methionine (20 mM) prepared in 100 mM tris buffer, pH 8. 3) Triton-X-100 (1 %, vv) in 100 mM tris buffer, pH 8. 4) Hydroxylamine hydrochloride (10 mM) prepared 100 mM tris buffer, pH 8. 5) Ethylenediamine tetraacetic acid (EDTA, 100 PM) prepared in 100 mM tris buffer, pH 8. 6) Riboflavin (50 PM) prepared in 100 mM tris buffer, pH 8. 7) Orthophosphoric acid (5%, vv) in distilled water. 8) Sulfanilamide (1%, w/v) in orthophosphoric acid (5%, vv). 9) N- (1-naphthyl) ethylenediamine (0.1%, w/v) in distilled water. 10) Greiss reagent: Freshly prepared mixture of sulfanilamide (1%) and N- (1-naphthyl) ethylenediamine (0.1%) in the ratio 1:1 (vv). 11) Cocktail: Freshly prepared mixture of the reagents 1-5 in specified proportions as mentioned below.

	Components		Volume per tube (ml)
	Tris buffer, 100 mM pH 8		1.110
	L-methionine (20 mM)		0.075
	Triton-X-100 (1%)		0.040
	Hydroxylamine hydrochloride (10 mM)		0.075
	EDTA (100 PM)		0.100
	Total		1.400 ml

Procedure: The assay was performed in dark since the reaction itself is light dependent. Blank tubes, internal control and sample tubes were assigned in duplicates as follows:

Blank----------------cocktail (1.40 ml) + buffer (0.18 ml)

Internal control----cocktail (1.40 ml) + buffer (0.1 ml) + riboflavin (0.08 ml)

Sample--------------cocktail (1.40 ml) + sample (0.1 ml) + riboflavin (0.08 ml)

The assay mixture (1.58 ml) contained hydroxylamine hydrochloride (0.47 mM), L-methionine (0.9 mM), EDTA (6.33 mM), triton-X 100 (0.026%), in 100 mM tris buffer, pH 8 and/or sample (0.1 ml containing 50 -150 µg protein) and riboflavin (2.5 µM). The tubes were vortexed and the reaction was started by exposing it to two 20 W fluorescent lamps (separated by 30 cm, fitted parallel to each other inside an aluminum coated wooden chamber) for 10 mins at 25 ºC. After 10' exposure to light, Greiss reagent (1 ml) was added to all tubes and mixed thoroughly. Absorbance of internal control and various sample tubes were read against blank at 540 nm in a UV-VIS spectrophotometer (Cary 100, Varian). One unit of enzyme activity was calculated from the value (Vo/V) 1 (Vo= absorbance of internal control, V=absorbance of sample). Final SOD activity was calculated by subtracting the value obtained for boiled samples (95 ºC for 30 min) from the corresponding unboiled samples. Enzyme activity was expressed as Units/mg protein.

Catalase

Principle: Catalase (CAT, EC 1.11.1.6) breakdown of H2O2 to water and oxygen. The enzyme activity was measured in the samples by monitoring the decrease in absorbance of H2O2 at 240 nm.

Reagents: 1) Potassium phosphate buffer, pH 7.0 (50 mM). 2) Absolute ethanol Triton-X-100 (10%, vv), 3) Hydrogen peroxide (H2O2, 25 mM) prepared in potassium phosphate buffer, pH 7.0 (50 mM).

Procedure: Prior to the assay, to 0.5 ml of PMF, 5 µl ethanol (1%) was added to prevent formation of the inactive complex of CAT (complex-II) by reacting with H2O2. This was kept in ice for 30'. After incubation, 50 µl triton-X 100 (1%, v/v) was added and incubated for 15' to increase the observable CAT activity by releasing it from peroxisomes (Cohen et al., 1970). Freshly prepared H2O2 (25 mM) in 2.9 ml PB (50 mM, pH 7) was pipetted into glass test tubes kept in water bath at 25 qC. The above treated PMF sample (0.1 ml) appropriately diluted so as to contain 50-200 Pg protein was then added to the tube. The solution was quickly transferred to quartz cuvette for measurement. Enzyme activity was calculated from the extinction coefficient of H2O2 as 43.6 M-1cm-1 and was expressed as nKat per mg protein. (One Kat is defined as one mole of H2O2 consumed per second per mg protein).