TITLE: 15 Determination of Enzyme Activity
AUTHORS: TJUSLS China

[STEPS]
?. [System setup – protein concentration]
Soak the 96-well plates in 75% ethanol and put the container in ultrasonic cleaner for 30min to 1 hour, then use ddH2O to wash these plates several times. Put these clean plates in drying oven at 55°C. ~
55 °C
?. [System setup – protein concentration]
Dilute the enzyme using its buffer. There we pipet 1 μL protein stock solution in 1mL buffer and mix gently. Then pipet 100 μL protein solution and mix with 400μL buffer each time, in order to dilute it as a 5-time gradient.
?. [System setup – protein concentration]
Pipet 94μL protein solution into 9 wells in plate, usually choosing B2-D4 area, to set 3 parallel controls. Pipet 94 μL buffer without protein into 3 wells as negative controls. Then add 6μL fluorescent substrate into wells.
?. [System setup – protein concentration]
Set up the program in Infinite M1000 Pro Automatic Microplate Reader. Shake for 10 sec at 654 rpm.Kinetic Cycle (to read fluorescent intensity each cycle)Fluorescent measure, 75 cycle, 10sec for each cycle.
?. [System setup – protein concentration]
Put the plate in Microplate reader, and click Start button.
?. [System setup – protein concentration]
When the facility ends testing, save data and import it into GraphPad Prism Software. Use “nonlinear fit” – “straight line” and compare R2 of lines under different concentrations to pick up the best linear fit one, whose R2 is most close to 1 .
?. [System setup – protein concentration]
Take this concentration as standard value, then set up parallel gradient of its 2x, 0.5x, 0.25x, etc. Repeat step 3-5.
?. [System setup – protein concentration]
Calculate the ratio of emission(rE = Q0/Qm, Q0 means the maximum fluorescent intensity of each reaction under different protein concentrations, Qm means the maximum fluorescent intensity of all reactions under different protein concentrations). Use GraphPad Prism Software to calculate EC80 value. Set log(concentration of protein) as X, the rate of emissionas Y. Use “nonlinear fit” – “log(agonist) vs. response—Find ECanything”, input 80 as the value of F parameter.
?. [System setup – protein concentration]
Usually we use the EC80 value as suitable protein concentration, and it can be adjusted according to the actual situation.
?. [System setup – buffer]
Design experimental groups with the “N+(N-1) principle”. Since we use PBS as our protein buffer, and class B beta-lactamases are depend on Zn2+, so we choose the concentration of NaCl, the concentration of ZnCl2, and pH, as variables.
?. [System setup – buffer]
Repeat step 3-5 to measure.
?. [System setup – buffer]
When the facility ends testing, save data and import it into GraphPad Prism Software. Use “nonlinear fit” – “straight line” to calculate the initial velocity of each reaction a.k.a. its slope value. Choose the condition with higher initial velocity.
?. [Kinetic Constant Measurement]
Dilute protein again with the ensured most suitable solution into proper concentration.
?. [Kinetic Constant Measurement]
Dilute the fluorescent substrate as 2-time gradient for 8 groups.
?. [Kinetic Constant Measurement]
Repeat step 3-5 to measure.
?. [Kinetic Constant Measurement]
When the facility ends testing, save data and import it into GraphPad Prism Software. Use “nonlinear fit” – “straight line”  to calculate the initial velocity of each reaction a.k.a. its slope value.
?. [Kinetic Constant Measurement]
Use “nonlinear fit” – “Michaelis-Menten” to fit Michaelis plot of this beta-lactamase. At the same time the software will calculate kinetic constants Km, Vmax automatically.
?. [Kinetic Constant Measurement]
Dilute protein as 2-time gradient for several groups. Repeat step 3-5 to measure. Take FI as Y, [S] as X, then use “nonlinear fit” – “straight line” to calculate fluorescent calibration value.
?. [Kinetic Constant Measurement]
Calculate kcat value. Kcat = Vmax/[E].