Enzyme Inhibition Test Enzyme Inhibition Test

------Inhibition of Succinate Dehydrogenase by Inhibition of Succinate Dehydrogenase by Oxalate

PURPOSE PURPOSE

To understand the principles of enzyme inhibition

To understand how to detect enzyme inhibition

To understand the clinical significance of enzyme inhibition

Competitive inhibition

•Competitive inhibitors usually resemble the substrate

•S and I “compete” for the enzyme active site

•The effect of a competitive inhibitor can be overcome with high concentrations of the substrate.

•The relative concentration of The relative concentration of substrate and inhibitor and their substrate and inhibitor and their respective affinityrespective affinity with the enzyme with the enzyme determines thedetermines the degree of degree of competitive inhibition. competitive inhibition.

Clinical significance of enzyme inhibitionClinical significance of enzyme inhibition

The usefulness of the most important pharmaceutical agents, antimetabolites, is based on the concept of competitive enzyme inhibition. – The antimetabolites are structural analogues of n

ormal biochemical compounds. – As competitive inhibitors they compete with the

naturally substrate for the active site of enzyme and block the formation of undesirable metabolic products in the body.

Succinate Dehydrogenase (SDH)

Succinate Dehydrogenase

FAD FADH2

HOOC CH

CH COOH

Fumarate

H

H COOHCH

CH COOH

Succinate

•This enzyme coupled with the coenzyme FAD is bound to the inner membrane of the mitochondria.

•The enzymatic activity of succinate dehydrogenase can be measured by monitoring the reduction of an artificial electron acceptor, methylene blue (MB). •The oxidized form of the dye (MB) is blue and the reduced form (MBH2) is colorless.

Fumarate

succinate dehydrogenase

MB(blue)

MBH2

(colorless)

FADH2

FAD

COO

COOOxalate

FAD FADH2

Competitive inhibition

•In the reaction succinate → fumarate, oxalate (or malonate) has a structure similar to succinate and competes with it for the active site on succinate dehydrogenase.

Reagents and MaterialsReagents and Materials

1. Phosphate buffer (pH 7.4) or 0.9% NaCl (NS,normal saline)

2. 0.25% Succinate solution3. 0.5% Oxalate solution4. Methylene blue (MB)5. Mitochondria preparation: SDH is bound to th

e inner membrane of the mitochondria. Rat liver is an ideal source for functional intact mitochondria. We use rats for our studies.

PROCEDUREPROCEDURE

A. Preparation of Mitochondria from Rat Liver

(1) Cervical dislocation Press the head of the rat with the left thumb

and forefingers. Grasp the base of the tail with the right thumb and forefingers and with a quick motion pull away and slightly upward from the skull.

This should cause a separation of the spinal cord from the brain.

liver

•A medial incision (vertical, up the middle of the anima) from groin to sternum by scissors and tweezers, first separating the skin then the muscle and peritoneum, reveals the liver, which is dark brown and large.•Drop the liver in the mortar, cut it into pieces, grind by the pestle for homogenizing. Then add phosphate buffer 7.0ml (in batches, maybe 2+2+3ml) and grind after adding phosphate buffer each time each time.•Transfer the homogenate to a Centrifugal tube, centrifuge at 3,000 rpm (revolutions per minute) for 3 minutes.

(2) Preparation of Mitochondria

•Transfer the supernatant to another clean tube, which contains mitochondria .

Centrifugation Centrifugation

Centrifugation is a process that involves the use of the centrifugal force for the separation of mixtures.

More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis.

Centrifuge (<6,000rpm)

Ultracentrifuge(>30,000rpm)

Microcentrifuge(<25,000rpm)

rpm- revolutions per minute

Notice for centrifugationNotice for centrifugation

•The liquid added to the centrifugal tubes should be even. Large difference may cause big shaking when in running state. •The tubes should be placed symmetrically by even number.

B. Label the test tubes according to B. Label the test tubes according to following table and add the reagentsfollowing table and add the reagents

Reagents 1 2 3 4 5

0.25 % Succinate (ml) 0.50 --- 0.50 2.00 0.50

0.5% Oxalate (ml) --- 0.50 0.50 0.50 2.00

dH2O (ml) 2.00 2.00 1.50 --- ---

Mitochondrial preparation (ml)

1.00 1.00 1.00 1.00 1.00

MB 5 drops 5 drops 5 drops 5 drops 5 drops

• Mix the reagents in each tube and incubate at 37C (no shaking!). Look at disappearance of the blue color in each tube and record its turn and time, then analyze.