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Some properties of an enzyme a demonstration experiment
48
Summer 1973 Vol. 1 No. 3
BIOCHEMICAL EDUCATION
i
ii
i
D.F. EVERED
SOME PROPERTIES OF AN ENZYME A DEMONSTRATION EXPERIMENT
Department of Biochemistry, Chelsea College (University o f L o n d o n ) , Manresa R o a d , Chelsea,
The requirements of a demonstration experiment are that it should be visible from the back row of a large lecture theatre. It should use cheap, readily available materials and apparatus. Furthermore, it should always work. If an experiment is to be repeated by students it should be open-ended to suggest additional experiments. The present experiment illustrates the catalytic action of the hydrolytic enzyme, alkaline phosphatase, utilising phenolphthalein diphosphate as substrate. Hydrolysis of this substrate releases phenolphthalein which produces a red colour in the alkaline medium. This colouration can be seen by eye and need not be measured by a photoelectric spectrophotometer.
L o n d o n SW3 6 L X
RESULTS It should be found that the dialysis bags in tubes 2 and 4 are fully coloured. The bag in tube 5 is colourless. Tubes 1 and 3 show bags which have a diminished coloration. With a stored or partially-hydrolysed sample of substrate magnesium phosphate may precipitate in tube 2. CONCLUSIONS These experiments demonstrate that:
REAGENTS
1. Enzymes are macromolecules and, therefore, cannot pass through a dialysis bag.
BUFFER, pH 10, 5.8g Na2CO3 and 3.8g NaHCO3 dissolved in water to 1 litre. ENZYME, alkaline phosphatase solution, freshly prepared, 10 mg in. 10 ml buffer. Commerical enzyme is suitable (British Drug Houses Ltd., Poole, Dorset). ENZYME AND INHIBITOR, alkaline phosphatase solution containing O.2M inhibitor, 4 ml enzyme solution containing 240 mg sodium citrate. SUBSTRATE, phenolphthalein diphosphate solution, O.1M (Sigma (London) Chemical Co. Ltd., London, S.W.6) freshly prepare 280 mg in 5 ml buffer. Extract any free phenolphthalein with an equal volume of ethyl acetate with gentle mixing. Discard the ethyl acetate layer using a teat pipette. ACTIVATOR, magnesium acetate solution, 2M, 2g tetrahydrate in 5 ml buffer.
2. The catalytic action of the enzyme is destroyed by heating. 3. Substrates and metal ion activators are mostly small molecules that can pass through dialysis tubing. DISCUSSION The present experiment is unusual in that the inhibitor, citrate, exerts its inhibitory effect competitively with the metal ion activator, Mg 2 + , and not the substrate. The inhibition is removed by adding Mg2÷ This effect has been shown with alkaline phosphatase from human blood serum (Steenson and Evered, 1963) and calf small intestine (Evered and Steenson, 1964). Furthermore, the reaction has been used as an assay of alkaline phosphatase in blood serum in medical laboratories (Huggins and Talalay, 1945).
INHIBITOR, sodium citrate solution, 2M, 3g in 5 ml buffer. SUGGESTED EXPERIMENTS METHOD
1. Determine the pH optimum of the enzyme by using buffers of different known pH values. With acidic buffers it will be necessary to add alkali to the contents of the dialysis bags at the end of the experiment.
Set up five large test tubes (24 x 150 mm) as in Table.
THE DEMONSTRATION OF ALKALINE PHOSPHATASE ACTIVITY Tube Number Addition 1
None
2
Activator
3
Inhibitor
4
Inhibitor and Activator
5
Boiled enzyme (Control)
2. Study the specificity of different metal ions for activating the enzyme e.g. substitute Zn 2÷, Co 2÷, etc. for the Mg2÷ ions (Clark and Porteous, 1965).
Buffer Activator Inhibitor Dialysis ml ml ml Bag ml 10
REFERENCES
-
-
2 enzyme
9
1
-
2 enzyme
1. Clark, B. and Porteous, J.W. (1965) Bioehem.J. 9 5 , 4 7 5 - 4 8 2 .
9
-
1
2 ml enzyme + inhibitor
2. Evered, D.F. and Steenson, T.I. (1964) Nature, Lond. 202, 491-492.
8
1
1
2 ml enzyme + inhibitor
3. Huggins, C. and Talalay, P. (1945). J.biol.Chem. 159, 399-410.
10
-
-
2 ml enzyme after boiling*
4. Steenson, T.I. and Evered, D.F. (1963) Lancet, ii, 792.
*The rest of the enzyme solution, after samples are taken for tubes 1 and 2, is placed in a dialysis bag closed with double knots at each end. The bag is placed in a boiling water bath for 10 min., cooled and then put into tube 5.
ACKNOWLEDGEMENT My thanks axe due to Miss Norunn Persson and Mr. J.F. Barley for skilled technical assistance,
Summer 1973 Vol. 1 No. 3
BIOCHEMICAL EDUCATION
i
ii
i
D.F. EVERED
SOME PROPERTIES OF AN ENZYME A DEMONSTRATION EXPERIMENT
Department of Biochemistry, Chelsea College (University o f L o n d o n ) , Manresa R o a d , Chelsea,
The requirements of a demonstration experiment are that it should be visible from the back row of a large lecture theatre. It should use cheap, readily available materials and apparatus. Furthermore, it should always work. If an experiment is to be repeated by students it should be open-ended to suggest additional experiments. The present experiment illustrates the catalytic action of the hydrolytic enzyme, alkaline phosphatase, utilising phenolphthalein diphosphate as substrate. Hydrolysis of this substrate releases phenolphthalein which produces a red colour in the alkaline medium. This colouration can be seen by eye and need not be measured by a photoelectric spectrophotometer.
L o n d o n SW3 6 L X
RESULTS It should be found that the dialysis bags in tubes 2 and 4 are fully coloured. The bag in tube 5 is colourless. Tubes 1 and 3 show bags which have a diminished coloration. With a stored or partially-hydrolysed sample of substrate magnesium phosphate may precipitate in tube 2. CONCLUSIONS These experiments demonstrate that:
REAGENTS
1. Enzymes are macromolecules and, therefore, cannot pass through a dialysis bag.
BUFFER, pH 10, 5.8g Na2CO3 and 3.8g NaHCO3 dissolved in water to 1 litre. ENZYME, alkaline phosphatase solution, freshly prepared, 10 mg in. 10 ml buffer. Commerical enzyme is suitable (British Drug Houses Ltd., Poole, Dorset). ENZYME AND INHIBITOR, alkaline phosphatase solution containing O.2M inhibitor, 4 ml enzyme solution containing 240 mg sodium citrate. SUBSTRATE, phenolphthalein diphosphate solution, O.1M (Sigma (London) Chemical Co. Ltd., London, S.W.6) freshly prepare 280 mg in 5 ml buffer. Extract any free phenolphthalein with an equal volume of ethyl acetate with gentle mixing. Discard the ethyl acetate layer using a teat pipette. ACTIVATOR, magnesium acetate solution, 2M, 2g tetrahydrate in 5 ml buffer.
2. The catalytic action of the enzyme is destroyed by heating. 3. Substrates and metal ion activators are mostly small molecules that can pass through dialysis tubing. DISCUSSION The present experiment is unusual in that the inhibitor, citrate, exerts its inhibitory effect competitively with the metal ion activator, Mg 2 + , and not the substrate. The inhibition is removed by adding Mg2÷ This effect has been shown with alkaline phosphatase from human blood serum (Steenson and Evered, 1963) and calf small intestine (Evered and Steenson, 1964). Furthermore, the reaction has been used as an assay of alkaline phosphatase in blood serum in medical laboratories (Huggins and Talalay, 1945).
INHIBITOR, sodium citrate solution, 2M, 3g in 5 ml buffer. SUGGESTED EXPERIMENTS METHOD
1. Determine the pH optimum of the enzyme by using buffers of different known pH values. With acidic buffers it will be necessary to add alkali to the contents of the dialysis bags at the end of the experiment.
Set up five large test tubes (24 x 150 mm) as in Table.
THE DEMONSTRATION OF ALKALINE PHOSPHATASE ACTIVITY Tube Number Addition 1
None
2
Activator
3
Inhibitor
4
Inhibitor and Activator
5
Boiled enzyme (Control)
2. Study the specificity of different metal ions for activating the enzyme e.g. substitute Zn 2÷, Co 2÷, etc. for the Mg2÷ ions (Clark and Porteous, 1965).
Buffer Activator Inhibitor Dialysis ml ml ml Bag ml 10
REFERENCES
-
-
2 enzyme
9
1
-
2 enzyme
1. Clark, B. and Porteous, J.W. (1965) Bioehem.J. 9 5 , 4 7 5 - 4 8 2 .
9
-
1
2 ml enzyme + inhibitor
2. Evered, D.F. and Steenson, T.I. (1964) Nature, Lond. 202, 491-492.
8
1
1
2 ml enzyme + inhibitor
3. Huggins, C. and Talalay, P. (1945). J.biol.Chem. 159, 399-410.
10
-
-
2 ml enzyme after boiling*
4. Steenson, T.I. and Evered, D.F. (1963) Lancet, ii, 792.
*The rest of the enzyme solution, after samples are taken for tubes 1 and 2, is placed in a dialysis bag closed with double knots at each end. The bag is placed in a boiling water bath for 10 min., cooled and then put into tube 5.
ACKNOWLEDGEMENT My thanks axe due to Miss Norunn Persson and Mr. J.F. Barley for skilled technical assistance,