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Effect of competitive and non–competitive inhibitors on ‑ galactosidase Paul Beaumont/Kath Crawford Gordon Moore/Anne Adams Science & Plants for Schools
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-galactosidase
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Competitive inhibition
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OR
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Non-competitive inhibition
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Non-competitive Inhibition
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Effect of [substrate] Competitive inhibitor – Increasing [substrate] displaces inhibitor from active site Non-competitive inhibitor – Increasing [substrate] has little or no effect on enzyme activity
![Effect of [substrate] Competitive inhibitor – Increasing [substrate] displaces inhibitor from active site Non-competitive inhibitor – Increasing [substrate] has little or no effect on enzyme activity](http://images.slideplayer.com/9/2546290/slides/slide_8.jpg "Effect of [substrate] Competitive inhibitor – Increasing [substrate] displaces inhibitor from active site Non-competitive inhibitor – Increasing [substrate] has little or no effect on enzyme activity")
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Method Carry out reaction – Without inhibitor at low [S] (ONPG) – In presence of inhibitor Galactose Iodine [I] chosen to completely inhibit reaction and look at increasing [S]
![Method Carry out reaction – Without inhibitor at low [S] (ONPG) – In presence of inhibitor Galactose Iodine [I] chosen to completely inhibit reaction and look at increasing [S]](http://images.slideplayer.com/9/2546290/slides/slide_9.jpg "Method Carry out reaction – Without inhibitor at low [S] (ONPG) – In presence of inhibitor Galactose Iodine [I] chosen to completely inhibit reaction and look at increasing [S]")
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Method steps 1-5 Dilute β-galactosidase (enzyme) Dilute stock ONPG (substrate) Mix diluted buffer and diluted ONPG in cuvette, zero colorimeter Add diluted enzyme to cuvette Read absorbance after two minutes
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Colorimeter R = ReferenceT = Test Direction of Beam
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Cuvette no 20% galactose in buffer (cm 3 ) ONPG stock solution cm 3 ) buffer (cm 3 )* ONPG x 20 dilution (cm 3 ) A reading [ONPG] in cuvette 12--1.04.3 x 10 -4 220.250.75-2.15 x 10 -3 320.5 -4.3 x 10 -3 420.750.25-6.45 x 10 -3 521.00-8.6 x 10 -3
![Cuvette no 20% galactose in buffer (cm 3 ) ONPG stock solution cm 3 ) buffer (cm 3 )* ONPG x 20 dilution (cm 3 ) A reading [ONPG] in cuvette x x x x x 10 -3](http://images.slideplayer.com/9/2546290/slides/slide_12.jpg "Cuvette no 20% galactose in buffer (cm 3 ) ONPG stock solution cm 3 ) buffer (cm 3 )* ONPG x 20 dilution (cm 3 ) A reading [ONPG] in cuvette x x x x x 10 -3")
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I = Galactose steps 6-7 Prepare cuvettes Each contains 2 cm 3 galactose (I) Cuvettes 1 – 6 contain increasing [S] NOTE – diluted (x 20) ONPG into tube 1, ONPG stock and buffer into tubes 2 – 6 Mix, cuvette in colorimeter, zero colorimeter Add 0.5 cm 3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min
![I = Galactose steps 6-7 Prepare cuvettes Each contains 2 cm 3 galactose (I) Cuvettes 1 – 6 contain increasing [S] NOTE – diluted (x 20) ONPG into tube 1, ONPG stock and buffer into tubes 2 – 6 Mix, cuvette in colorimeter, zero colorimeter Add 0.5 cm 3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min](http://images.slideplayer.com/9/2546290/slides/slide_13.jpg "I = Galactose steps 6-7 Prepare cuvettes Each contains 2 cm 3 galactose (I) Cuvettes 1 – 6 contain increasing [S] NOTE – diluted (x 20) ONPG into tube 1, ONPG stock and buffer into tubes 2 – 6 Mix, cuvette in colorimeter, zero colorimeter Add 0.5 cm 3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min")
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I = Iodine steps 8-9 Prepare cuvettes Each contains 1 cm 3 iodine (I) Cuvettes 1 – 3 contain increasing [S] NOTE – diluted (x 20) ONPG into tube 1, ONPG stock and buffer into tubes 2 – 3 Mix, cuvette in colorimeter, zero colorimeter Add 0.5 cm 3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min
![I = Iodine steps 8-9 Prepare cuvettes Each contains 1 cm 3 iodine (I) Cuvettes 1 – 3 contain increasing [S] NOTE – diluted (x 20) ONPG into tube 1, ONPG stock and buffer into tubes 2 – 3 Mix, cuvette in colorimeter, zero colorimeter Add 0.5 cm 3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min](http://images.slideplayer.com/9/2546290/slides/slide_14.jpg "I = Iodine steps 8-9 Prepare cuvettes Each contains 1 cm 3 iodine (I) Cuvettes 1 – 3 contain increasing [S] NOTE – diluted (x 20) ONPG into tube 1, ONPG stock and buffer into tubes 2 – 3 Mix, cuvette in colorimeter, zero colorimeter Add 0.5 cm 3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min")
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Step 10 Add buffer and diluted (x 20) ONPG to cuvette, mix and zero colorimeter Add 0.5 cm 3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min Compare with results after step 5.
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Results
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Results
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Results
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