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This paper will look at why three topics that concern the calcium and proton activities in rat cardiac mitochondria. The three topics include; composition of the ionic milieu within the mitochondrial matrix is a controversial issue, quantification of the matrix in isolated mammalian mitochondria, as well as how the application of the fluorescence of spectroscopy to research on mitochondrial function poses peculiar problems to the organelle and their experimental evidences.

Why the composition of the ionic milieu within the mitochondrial matrix is controversial.

The composition of the ionic milieu of mitochondrial matrix especially within the mammals remains a controversial issue, despite the fact that fura-2 and 2’, 7’ –bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, fluorescent probes of {ca2+} and {H+} have been efficaciously loaded into the mitochondria of rats (Davis et al, 123). This is because there are physiological fluctuations that are influenced by the calcium content of the mitochondria that provoke changes, which in turn control the, milieu dehydrogenases. This therefore implies that the free calcium cations are controlled by the uptake and discharge of the calcium cation across the plasma membrane, the inner membrane of the mitochondria, as well as the endoplasmic reticulum. Homeostasis plays a role here because each of these three membranes has their own distinct transport systems which regulate the calcium cations that are present the cells.

Experimental evidence

An experiment was carried out to continuously observe the matrix that was free of the concentration of the Ca2+ of the isolated heart mitochondria of rats. Fluorescent calcium cations indicators were used, i.e. fura-2 and quin 2. The mitochondria that had been isolated hydrolyzed the indicators. The results were that when the calcium content of the mitochondria was increased, there was a corresponding rise in the calcium cations (Ishiyama et al, 1518). These results therefore indicate that the physiological variations in the calcium content of the mitochondria were responsible for the prompting of the calcium cations that in turn regulated the dehydrogenases of the matrix. The matrix dehydrogenase is the enzyme present in the matrix that transfers the hydrogen ions.

Why Quantification of matrix in isolated mammalian mitochondria is problematic.

The quantification of the matrix in the isolated mitochondria [Ca2+] of mammals is a problem. This is evidenced by the fact that current evaluations of the calcium cations in the mitochondria of generally respiring organelles range from 30nM to 1mM. Mitochondria play a major role in the regulation of the maintenance of the Ca2+ which have a role in homeostasis as well, as the body responds to normal bodily stimuli. Furthermore, mitochondria play a role in certain pathophysiological environments.

Experimental evidence

An experiment was carried out using fluorescent dyes that had a high specificity for ions , especially calcium cations [Ca2+] and  hydrogen ions [H+]. These dyes could be effectively and rapidly loaded with very little effect on the viability of the cell as well as the Trans membrane ion gradients. Initially, the Ca2+ fluorescent dye could not collect into the intracellular organelles. It was however difficult even with the development of more fluorescent dyes, to quantify either of the matrix i.e. [Ca2+] or [H+]. This was due to the presence of the fluorescent dyes which were partially hydrolyzed, making them more complex, as well as the presence of the free fluorescent dyes that were sensitive to ions (Haas, 145). The situation became more complex especially with the accumulation of the several species of the dye both in and out of the mitochondrial addition, the complexities came about because of the leakage of the fluorescent dyes form the organelles that were being examined. This presupposes that there is need for the correction of the accumulation of the dye that collects outside the organelle, which poses even more difficulties.

The application of fluorescent spectroscopy to research on mitochondrial function and the problem it poses

The application of fluorescence spectroscopy to research on the function of the mitochondria poses challenges that are specific to the mitochondria. There have been no experiments that have been carried out to establish the affinities of the fluorescent dyes to ion. For example, there have been no spectral properties that have ever been carried out beyond the PH of 8 but chances are that the binding affinity of the hydrogen ions is likely to change over the range of calcium cations that are thought to exist within the matrix of the mitochondria Conley et al, 335).

Experimental evidence

The fluorescent dye fura-2 was put into the mitochondria and the mitochondria disabled but in such a way that it would allow for the superfusion of the organelles of the mitochondria. The mitochondrion was then observed under a fluorescence microscope and the calcium cations were found to be low even when the mitochondria had been isolated using standard techniques even at superfluous buffers of the mitochondrion. This shows that the quantification of the calcium cations as well as the hydrogen ions is difficult because their low concentration and this can be attributed to the super fusion of the organelles.


The composition of the ionic milieu of within the mitochondrial matrix of a mammal is controversial because of the many functions of the mitochondria for example homeostasis, and the fluctuations in the calcium cations, and also because the membrane of the mitochondrion is dependent on the maintenance of the hydrogen ions. The quantification of the matrix in isolated mammalian mitochondria is difficult as well. This is majorly because of the homeostatic mechanisms that determine mitochondrial matrix ion content. The last topic of the problem that the application of the fluorescence spectroscopy poses problems that is very specific to the mitochondria itself. For example the high rate of surface area of the membrane to the volume of the matrix when it is compared to most of the other cells.  

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