The Effect Of Malonate Concentrations On Cellular Respiration

Introduction

At the most fundamental level, cellular respiration is the process which is responsible for keeping organisms alive. It is the biochemical means whereby food is transformed in to the form of energy that a cell requires. This type of energy is called Adenosine Triphosphate (ATP) and it is the fuel that every cell utilizes to maintain its health, growth and its ability to reproduce.

In eukaryotes, cellular respiration as a whole can be broken down further into three metabolic processes; Glycolisis, which occurs in the cytoplasm of a cell; The Krebs Cycle which takes place in the mitochondrial matrix; and The Electron Transport Chain which occurs within the mitochondrial membrane. This experiment will focus on a particular step which occurs within The Krebs Cycle whereby the four carbon molecule succinate is oxidized by the enzyme succinate dehydrogenase to transform it into another four carbon molecule called fumerate. It has been established that malonate can act as a competitive inhibitor to succinate in this process due to its ability to bind in the place of succinate in succinate deydrogenase. A substance called di-chorophenol-indophenol (DCPIP) will be added to each sample as a means to measure the level of oxidation which has occurred. DCPIP will turn from blue to colourless in the event that cellular respiration is occurring. Transmittance percentages will be calculated using a spectrophotometer. This will give a quantified result of the cellular respiration rate at each time point. The endeavor of this experiment was to measure what affect various concentrations of malonate had on the respiration rate of Phaseous lunatus (P. lunatus) mitochondria. It was hypothesized that an increased concentration of malonate would result in a decrease in the cellular respiration of P. lunatus mitochondria.

Methods

Methods were adapted from Flinders University (2019). A spectrophotometer was used to measure the transmittance of each sample at 600nm. Each cuvette had a total volume of 3 mL. The inhibitor amount was varied for each sample. Malonate was used as the inhibitor. The buffer, DCPIP, succinate and malonate were measured into cuvettes labelled B (blank), Control, 1, 2 and 3. Amounts of each constituent were measured. The mitochondrial suspension of P. lunatus was added just prior to taking the first reading at zero time point. The mitochondrial suspension was added to the cuvette labelled B. Parafilm was used to cover the cuvette tightly and it was gently inverted to disperse the contents evenly. The timer was started. The blank was then placed into the spectrophotometer and it was zeroed to give a transmittance of 100%. This process was repeated for the Control and for Samples 1, 2 and 3. The mitochondrial suspensions were added to the controls and samples at the 1, 2, 3 and 4 minute respective points on the timer to assist with accuracy in readings. The transmittance percentages for the control and each sample were recorded at 5 minute intervals for a total of 40 minutes. Prior to the transmittance been read for each sample, each cuvette was gently inverted and the spectrophotometer was zeroed using the Blank. Six individual groups undertook the experiment. A total of 36 readings were taken per group. The mean results were calculated using Microsoft Excel and depicted graphically.

Results

The rate of cellular respiration for the control appears to peak at approximately the 15 minute mark. When comparing the transmission percentage values for the control to the samples, the trend suggested that the addition of malonate in higher concentrations has the greatest decreasing effect on cellular respiration. At the 15 minute time point, the transmittance of sample 3 (containing the highest level of malonate) is 34.65% compared to sample 1 (containing no malonate) where the transmittance is 73.12%. It is interesting to note that the rate of cellular respiration of the control is consistently higher than that of sample 3 despite the control containing no added substrate (succinate). This further suggests that the addition of malonate has a significant effect on the rate of cellular respiration. The rate of cellular respiration for sample 2 containing 0.2mL of succinate and 0.05 mL of malonate appears relatively similar to the rate of cellular respiration for the control suggesting that at these volumes, the inhibiting effect of malonate is enough to counteract the promoting effect of the additional succinate. The addition of DCPIP allows the degree at which cellular respiration is occurring over time to be measured. Transmittance measured at 600nm as a percentage (%).

Discussion

The aim of this experiment was to determine whether the addition of malonate would have a decreasing effect on the cellular respiration rate of P. lunatus mitochondria. Based on previous research suggesting that malonate has the ability to act as a competitive inhibitor to succinate, these results were expected. The experiment supported the hypothesis that increase concentrations of malonate would have a decreasing effect on the respiration rate of the P. lunatus mitochondria. Limitations of this experiment include it being a relatively basic experiment with only 3 samples being compared to a control and time constraints. Greater accuracy could be ascertained by including samples of additional concentrations of malonate and a longer experiment time. An anomaly for the reading at the 15 minute time point for sample 3 also suggests an error in data collection. Reducing the number of participants preparing each sample and confining the spectrophotometer calculations to being read by one single person may act to control variance in the method and provide more consistent estimates of transmission percentage values.

Conclusion

Concentrations of malonate result in a decrease in the cellular respiration rate of P. lunatus mitochondria. One potential area where the results of this experiment could be further explored is in relation to the treatment of cancers. Deliberately endeavouring to decrease the cellular respiration rate of the mitochondria of a tumor cell through the use of malonate, may force cancer cells into apoptosis. Utilising a nanoparitcle delivery system which serves to only target cancerous cells might hold promise as an alternate to current chemotherapy treatments.