The Kinetics of Acid Phosphatase practical

Question 1

role of phosphatase in cells - a hydrolase

comes from sigma aldrich (EC 3.1.3.2),

Answer 1

dephophorylating substrate - used o hyrolyse the phosphate groups from basic substrates

Question 2

substrate used
P nitrophenol phosphate
PNPP

Answer 2

enzyme will cut phosphate group
enzyme will attrack phsphate group then release
enxyme binds on the active site - hydrolytic reaction
adds water across the bond
electrons jump around

PNP
p-nitrophenol, is yellow at alkaline pH, and its appearance can be detected at 405
nm using a spectrophotometer

Question 3

aim

Answer 3

to prove michallus menten approach

rectanglular hyperbola

as you change the concentration of substrate what happens to the initial rate =vary reaction temperature (RT 37 + 50 degrees)

Question 4

time course reactions

Answer 4

start an enzyme reaction = at time points take a small point out and stop the reaction
level of reaction goes down

small aliquots - small volume
from time course at regular intervals (3mins)

quenching = stop reaction with sodium hydroxide - denaturing enzyme

Question 5

buffer

Answer 5

50 ml of 15 mM sodium-citrate and 3 mM MgCl2 at pH 5.0

magnesium ions = cofactor divalent cation
part of active site of enzyme

Question 6

method

Answer 6

1. sonication to release it from the
   cell in which it is found.
   purified from other proteins and macromolecules in
   a series of chromatographic purification stages, such as ion-exchange-, gel filtration- or affinitychromatography

put pnpp + enzyme tubes in ice to keep cold
enzyme conc is still lower than lowest substrate conc

2. boiling tube will contain reaction:
   3.8ml buffer
   0,7ml substrate
   2ml quench naoh solin temp prelabelled test tubes with 2mls naoh prealiquoted

• pipette slowly and smooth as so not to asperate liquid
• changing tips each time

4. at the waterbath pippete 0.5ml of enzyme to tube + mix

set pipette to 0.6ml ready to aquilot out time 0

every 3 ins for 8

5. decant into cuvettes
6. 6ml
7. spectomotometry:

calibrate
set ref button to blank once with q0 in = control

repeat with all times and record

Question 7

conc

Answer 7

as u increase conc of substrate - reaches a Vmax 0.04