EXPERIMENT 2 COMPITITIVE INHIBITION OF MALONATE
TO SUCCINIC DEHYDROGENASE(SDH)
PRINCIPLE
SDH is an important dehydrogenase in human body. It catalyzes succinate into
fumarate by get off 2H. Its ligand is FAD. FAD can accept 2H from succinate and
become FAD·2H which can pass 2H through respiratory chain and form H2O by
combining 1/2 O2 in vivo. But in vitro, FAD·2H can deliver 2H to methylene blue.
Methylene is then reduced to methylene white.
CH2COOH
│
CH2COOH
HOOC-CH
‖
HC-COOH
FAD
Methylene white
FAD·2H
Methylene blue
Malonate is a structural analog of succinate. It can competitively combine with
SDH and occupy its active center. SDH cannot catalyze malonate’s dehydration.
Therefore, if the enzyme’s active center is occupied by malonate, the enzyme’s
activity will certainly decreased since SDH, at the same time, cannot work on
succinate. If the concentration of substrates is enhanced, the inhibition of malonate
may be accordingly decreased or eliminated.
PROCEDURE
1. Preparation of heart homogenate of pig.
Weigh 30gof pig’s cardiac muscle and snip with scissors. Put the muscle in a
grass homogenizer. Grind them for 3 minutes by adding 80ml of 0.1mol/L
phosphate buffer. Add 70ml more of the same buffer after taking out muscle
homogenate from the homogenizer and mix up.
2. Label 4 EXPERIMENT tubes and do as the following table.
(drops)
Tubes cardiac muscle 15g/L Sodium 10g/L Sodium distilled 0.2g/L methylene results
homogenate
succinate
malonate water
blue
1
10
10
—
20
5
2
10
10
10
10
5
3
—
10
10
20
5
1
4
10
20
10
—
5
Mix up after methylene blue is added. Then add 10 drops of paraffin along the
tube wall. Don’t shake after adding paraffin! Settles tubes down at room
temperature.
3. Observe and record the velocities, early or late and extent of fading in tubes. The
results will be obvious within 10~15minutes. Analyze the reasons of different
results in tubes.
REAGENTS
1. 15g/L Sodium succinate solution Weigh 1.5g sodium succinate and dissolve it
in distilled water. The final volume is 100ml(If prepare the solution with
succinate, you may firstly prepare 15g/L succinate solution. Then adjust it to
pH7~8 with 5mol/L NaOH).
2. 10g/L Sodium malonate solution Weigh 1.0g sodium malonate 1.0g and
dissolve it in distilled water. The final volume is 100ml(If prepare with malonate,
the method is the same as 1).
3. 0.2g/L Methylene blue.
4. 0.1mol/L Phosphate buffer(pH 7.4)(Please consult to appendix 1).
2