Bke2 Biochemistry Lectures

Lecture 17: Oxidative phosphorylation lab, summary of energy production in the cell
Reading material: Lab instructions, Horton chapters 12,13 & 15

Abstract: Mitochondria are the energy producing centres in the cell and are responsible for the conversion of chemically bound energy to ATP.

Most of the enzyme activities from different enzymes can be measured by using suspensions of disrupted cells or cell organells. To study ATP synthesis coupled to respiratory electron flow it is necessary to use intact mitochondria. The mitochondria are separated from the homogenate by sequential centrifugation. Isolated mitochondria are suspended in a buffer containing ADP, Pi and oxidisable substrate such as succinate. Three easily measured processes occur: 1) the substrate is oxidised, 2) O₂ is consumed and 3) ATP is synthesised.

In the laboratory experiment named oxidative phosphorylation, O₂ consumption is measured using a Clarks oxygen electrode. Oxygen consumption and ATP synthesis are dependent upon substrate oxidation and the ADP/O quotient is determined for three different substrates. Because the energy of substrate oxidation drives ATP synthesis in mitochondria, it is not surprising that inhibitors of the passage of electrons to O₂ block ATP synthesis. In the laboratory experiment, the effect on the respiratory chain of three different inhibitors will be studied.

For more details see wet Lab: Oxidative phosphorylation.

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