How to lyse mitochondria in order to measue complexIII activity? - (Jul/25/2008 )
Hello
I want to test the mitochondrial complex III (cytochrome bc1) activity. I am going to isolate mitochondria using a kit. After that, do I need to further disrupt the mitochondria to release the enzyme? If yes, anybody knows how to disrupt the mitochondria but maintain the enzyme activity? Thanks very much.
Jae
-flyingfish2000-
QUOTE (flyingfish2000 @ Jul 25 2008, 08:38 AM)
Hello
I want to test the mitochondrial complex III (cytochrome bc1) activity. I am going to isolate mitochondria using a kit. After that, do I need to further disrupt the mitochondria to release the enzyme? If yes, anybody knows how to disrupt the mitochondria but maintain the enzyme activity? Thanks very much.
Jae
I want to test the mitochondrial complex III (cytochrome bc1) activity. I am going to isolate mitochondria using a kit. After that, do I need to further disrupt the mitochondria to release the enzyme? If yes, anybody knows how to disrupt the mitochondria but maintain the enzyme activity? Thanks very much.
Jae
Complex III is transmembranous, with the cyt-c reactive site in the aqueous phase on the external (intermembrane) side and the ubiquinol-reactive sites within the membrane. Ubiquinol (or short-chains analogs used in the assay) is lipid-soluble so will reach its site. Cyt c is not membrane permeable. The outer membrane is pretty much gone in most preparations, so the real problem would be if some of the membrane is in inverted vesicles (submitochondrial particles; smp). Complex III in these vesicles will not react with externally added cyt c, so you would not get the full activity if the inner membrane is intact.
You can disrupt the inner membrane with nonionic detergents without damaging bc1 (in fact activity is stimulated by detergent, even in purified protein). About 1 g/L Triton X-100 or 0.1 g/L dodecyl maltoside should be OK if the protein is dilute; or 1 g detergent per g protein if that comes out to be more.
Ed
-eaberry-
Thank you very much, Ed.
-flyingfish2000-