IA on effect of inhibitor on the rate of hydrolysis of lactose (ONPG) by enzyme (lactase), measured using visible spectrophotometer.

1. Effect of inhibitor on the rate of hydrolysis of lactose by enzyme (lactase), measured using visible spectrophotometer.
Lactase enzyme from baby drops used. 10ul enzyme added to 1ml water as stock sol.
Lactose solution (ONPG) 5mM prepared, by dissolving 0.15g in 100ml water.
Yellow solution produce when enzyme breaks down ONPG (lactose)
Rate of hydrolysis is measure by change in abs over time
ONPG is lactose analogue which break down by enzyme lactase to produce
yellow substance which can be measure with colorimeter
Procedure:
500ul lactase was added to 500ul water, to make a stock enzyme solution.
50ul enzyme was added to 100ul of inhibitor (Pb, Cu, Co, Ni) conc - (1%w/v)
After incubating for 5 mins, 10ul of enzyme/inhibitor mix was added to 1ml of ONPG.
Both solution were added/mix into a cuvette and placed in colorimeter.
Change of abs over time (disappearance of yellow) – rate of hydrolysis.
Reagents needed
Mixing is crucial to ensure, expt work, it is advisable to add enzyme to the
bottom of cuvette and then pipette in ONPG (lactose) sol to mix them well

2. Go to expt – press calibrate Insert cuvette (yellow sol), press collect
Insert a blank containing water
Press stop, and click on rainbow icon. Select abs vs time. λ max at 391nm will
be automatically chosen
Abs vs time
Effect of inhibitor on the rate of hydrolysis of lactose by enzyme (lactase), measured using visible spectrophotometer.

3. 0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
Pb2+ Cu2+ Co2+ Ni2+ +ve control
Rate
of
hydrolysis
Type of inhibitors
Inhibitor vs rate of hydrolysis
Copper and nickel metal acts as best inhibitor on lactase enzyme
Change of abs over time were plotted.
Rate of hydrolysis – decrease in Abs over time
Slope/gradient taken over 6s
Inhibitor Rate
Abss-1
Pb2+ 0.01296
Cu2+ 0.0007684
Co2+ 0.00004078
Ni2+ 0.004687
+ve
control
0.00003544
Data collected.
Effect of inhibitor on the rate of hydrolysis of lactose by enzyme (lactase), measured using visible spectrophotometer.
+ve control – enzyme without any inhibitor