No method is perfect, unfortunately. Just to add to the list, you can normalize by compex I II III IV or V content. Or a total protein (for purified mitochondria it could be the best method, in my humble opinion). Any single enzyme activity can vary dramatically from sample to sample due to purification inconsistencies and individual differences in my opinion. Sorry for not givinig a definitive answer - there is no such thing. Just like in life in general. Things are often more complicated than they appear.

Hi Vadim, 

Thanks for the response. I have carried out quantification of protein using BCA assay therefore I load equal amounts of the mitochondria in each sample before examination. More so I have been measuring citrate synthase activity to normalise with alterations in the enzymes activity presenting during diabetes. But I guess, as you have stated, there is always going to be differences in opinion on normalising and who is correct or not.

Dear Micheal, normalization of the activities of OXPHOS enzymes activities using citrate synthase was introduced by Dr. Rustin. The activities are usually estimated  using submitochondrial particles. Rustin studied the enzymes in SMP prepared from cultured human lymphoblastoid cells of patients and found huge differences between individuals, which did not allowed the usage of any statistical method . So, he introduced  this normalization. He was completely wrong. The reason for huge  variance between patients was in the longevity and method of storage of either mitochondria or SMP. The enzymes simply underwent oxidative damage and because the samples were store for different times, the changes were also different. I studied this issue very carefully and found that citrate synthase is also undergoes oxidative damage. More information you can find in the attached excerpt from my book where I discuss SMP and measurements of OXPHOS activities. The book is free on my RG account. I have also to mention, that you cannot compare the quantity and activities of enzymes in different cultures of cells even of the same types. You can compare only qualitatively. There are many reasons for that.

Hi Alexander,

Thank you for the response, I have downloaded your excerpt and will read through it as I have some of your work on other mitochondrial topics. I find your pieces of work to be quite frank and offering a facts based opinion that differs with deemed 'conventional' methods being highly published and fascinating which allows for my greater critical analysis.

Thank you.