The Lysozyme: A model for Enzyme structure - function relationship. Experiment no. 1: Effect of temperature on enzymatic activity

Transcription

1 The Lysozyme: A model for Enzyme structure - function relationship Written by: Dr. Nurit Doron, Dr Michal Mendelovici, Revital Levi Edited by Dr Noa Ofrath Experiment no. : Effect of temperature on enzymatic activity Open the file: "lysozyme results" on the desk top and save it under your name in drive H. Label test tubes through. Add ml of 0.M phosphate buffer ( ph 6.) to each tube. Add 00µl of lysozyme solution (0.0mg/ml) to each tube. Vortex the tubes briefly. Temperature equilibration: Incubate each tube in the appropriate temperature (e.g. tube # in o C; tube # in 7 o C; tube # in 6 o C). 6. Turn on the spectrophotometer and allow it to warm up. Adjust the wavelength to 0 nm. 7. Place a tube with distilled water in the spectrophotometer and zero it (hit the blue button). In the next steps you are starting the enzymatic reaction. You will have to check enzymatic activity every minute for min. Each temperature will be checked separately Note: A. Work fast (you have minute intervals) B. Vortex (gently!) the reaction mixture before every reading C. Wipe well the tubes before reading D. Place back the tubes in the appropriate temperature in between readings 8. Take tube # out of the water bath and wipe it well 9. Mix well bacterial walls 0. Start your stopper. Add 00 µl bacterial walls to tube # ( o C).. Vortex the tubes briefly to ensure thorough mixing of the tubes content. Read immediately the OD by hitting the read button (green button) Mark the time!!. Write the reading in the table below (or on the computer). Quickly place the tube back in the water bath in the appropriate temperature

2 6. Repeat steps - in min intervals, up to minutes 7. Take tube # out of the water bath and wipe it well 8. Repeat steps 9-6 with tube # (7 o C) 9. Repeat steps 9-6 with tube # (6 o C) ABSORBANCE [OD (0) ] Tube # # temperature/ o C 7 C Time(min.) 0 # 6 C 8. Save your results in your computer file. A unit of lysozyme activity is defined as the amount of enzyme that will produce a DeltaA 0 of 0.00 per min at ph 6. at o C The official definition of an Enzyme Unit (EU) is the amount of enzyme that causes a change in OD0 of 0.00 absorbance units per minute (ph 7.0, oc).

3 Experiment no. : Effect of the reducing agent DTT on enzymatic activity Open the file: "lysozyme results" on the desk top and save it under your name in drive H. Label test tubes through. Add ml of 0.M phosphate buffer ph 6. to tubes,. Add ml of 0.M phosphate buffer ph 6. to tubes,. Add 00µl of lysozyme solution (0.0mg/ml) to each tube. Add to tubes and 000µl of DTT 6. Vortex the tubes briefly 7. Temperature equilibration: Incubate tubes no. & in the in o C and tube no. & in 6 o C. 8. Turn on the spectrophotometer and allow it to warm up. Adjust the wavelength to 0 nm. 9. Place a tube with distilled water in the spectrophotometer and hit the zero button (the blue button). In the next steps you are starting the enzymatic reaction. You will have to check enzymatic activity every minute for min. Each temperature will be checked separately Note: A. Work fast (you have minute intervals) B. Vortex (gently!) the reaction mixture before every reading C. Wipe well the tubes before reading D. Place back the tubes in the appropriate temperature in between readings 0. Take tube # out of the water bath and wipe it well. Mix well bacterial walls. Start your stopper. Add 00 µl bacterial walls to tube #. Vortex the tubes briefly to ensure thorough mixing of the tubes content. Read immediately the OD by hitting the "read" button (green button) Mark the time!! 6. Write the reading in the table below (or on the computer) 7. Quickly place the tube back in the water bath in the appropriate temperature 8. Repeat steps -7 in min intervals, up to minutes 9. Take tube # out of the water bath and wipe it well 0. Repeat steps -8 with tube #

4 . Repeat steps -8 with tube #. Repeat steps -8 with tube # ABSORBANCE [OD (0) ] Tube no. temperature o C 6 o C Time(min.)/ DTT Save your results on the computer

5 Experiment no. : Effect of an inhibitor (NAG) on enzymatic activity Open the file: "lysozyme results" on the desk top and save it under your name in drive H. Label test tubes through. Add ml of 0.M phosphate buffer ( ph 6.) to each tube. Add 00µl of lysozyme solution (0.0mg/ml) to each tube. Add 00µl of NAG 0. mg/ml to tube #. Change tip 6. Add 00µl of NAG mg/ml to tube # 7. Vortex the tubes briefly 8. Temperature equilibration: Incubate all the tubes in 7 o C 9. Turn on the spectrophotometer and allow it to warm up. Adjust the wavelength to 0 nm. 0. Place a tube with distilled water in the spectrophotometer and hit the zero button (the blue button). In the next steps you are starting the enzymatic reaction. You will have to check enzymatic activity every minute for min. Each temperature will be checked separately Note: A. Work fast (you have minute intervals) B. Vortex (gently!) the reaction mixture before every reading C. Wipe well the tubes before reading D. Place back the tubes in the appropriate temperature in between readings. Take tube # out of the water bath and wipe it well. Mix well bacterial walls. Start your stopper. Add 00 µl bacterial walls to tube # ( o C).. Vortex the tubes briefly to ensure thorough mixing of the tubes content 6. Read immediately the OD by hitting the read button (green button) Mark the time!! 7. Write the reading in the table below (or on the computer) 8. Quickly place the tube back in the water bath in the appropriate temperature 9. Repeat steps -8 in min intervals, up to minutes 0. Take tube # out of the water bath and wipe it well. Repeat steps -9 with tube #. Repeat steps -9 with tube #

6 Tube no. inhibitor/ Time(min.) 0 ABSORBANCE [OD (0) ] - NAG( 0. mg/ml) NAG ( mg/ml). Save your results on the computer 6