The membrane potential of mitochondria was estimated from the accumulation of tetraphenyl phosphonium (TPP+), which was determined with the TPP+-selective electrode developed in the present study. The preparation and some operational parameters of the electrode were described. The kinetics for uptake by mitochondria of TPP+ and DDA+ (dibenzyldimethyl ammonium) were analyzed, and it was found that TPP+ permeated the mitochondrial membrane about 15 times faster than DDA+. The final amounts of accumulation of TPP+ and DDA+ by mitochondria were approximately equal. For the state-4 mitochondria, the membrane potential was about 180 mV (interior negative). Simultaneous measurements of TPP+-uptake and oxygen consumption showed that the transition between states 3 and 4 was detectable by use of the TPP+-electrode. After the TPP+-electrode showed that state-4 was reached, the extra-mitochondrial phosphorylation potential was measured. The difference in pH across the membrane was measured from the distribution of permeant anion, acetate, so as to calculate the proton electrochemical potential. The ratio of extra-mitochondrial phosphorylation potential to proton electro-chemical potential, n was close to 3. This value of n was also found to be 3 when ATP was hydrolyzed under the condition that the respiratory chain was arrested. The implication that n = 3 was discussed.

We have developed a piezoelectric immunosensor for the detection of microalbumin. Human serum albumin (HSA) in the range 0.1-100 micrograms mL-1 could be detected using a flow cell; the immunosensor is sensitive enough to monitor levels of albuminuria. The immunosensor did not respond to bovine serum albumin, only to HSA, implying that the specificity for HSA was high. We investigated the relationship between the frequency change (delta F) and adsorption per unit area of piezoelectrically active quartz crystal (delta M). delta M was estimated with radioisotope-labeled anti-HSA or HSA. When anti-HSA was adsorbed onto the surface of the crystal or HSA was bound to anti-HSA supported by the crystal, values of magnitude of delta F/delta M were larger than the value predicted from theory (Sauerbrey's equation). Furthermore, magnitude of delta F/delta M for HSA was larger than that for anti-HSA.

No abstract

No abstract

No abstract