Question

This is a Biology lab about cellular respiration using mitochondrial suspension. Here's what my hypothesis is: An increased amount of the substrate succinate will increase the rate of cellular respiration.

I was sure that succinate would increase the rate of the reaction. But that was not what my graph showed. Here is what was put into each cuvette

Tube	Buffer	DCIP	Mitochondrial suspension	Succinate (add last)
1	4.4	0.3	0.3	0
2	4.3	0.3	0.3	0.1
3	4.2	0.3	0.3	0.2

Here's what my results are after measuring its light absorbance with a spectrophotometer

	Time (minutes)
Tube	0	5	10	15	20
1	0.438	0.412	0.406	0.412	0.408
2	0.400	0.374	0.356	0.343	0.331
3	0.390	0.359	0.333	0.316	0.301

Then i made this graph based on the table above

Redox reactions in a mitochondrial suspension Chart Area Absorbance (at 600nm) Time (minutes) . Series Series 2 Series 3

Can anybody help me interpret these results? I thought tube 3 (in gray) would be high up there since its has the most succinate in it, therefore the reaction would have the highest cellular respiration activity. Are my axis wrong? I asked my teacher but she didn't help, she just told me to think so I'm here seeing if anybody can explain this to me. I'm also wondering if what the spectrophotometer shows is the energy activation rate of the reaction. This would mean that the absorbance is inversely proportional to the rate of cellular respiration and its energy activation energy rate. So I don't know what to ultimately say in my conclusion part of the lab report and now I'm stuck. We cleaned the cuvettes with Kimwipes, had a tube to zero the spectrophotometer with that was made by the teacher. Also if someone think that the change in the buffer would affect this reaction more than adding succinate, please elaborate on that.

Hope somebody out there can help me. Please be specific, as you can see i overthink A LOT.

Thank you!

Answer 1

In your experiment, although you should keep the volume of the buffer the same but still we can interpret the result. Precisely the start point i.e 0min should have been the same but this is a real wet lab experiment limitation where we are not able to get an ideal start point. This is because adding the reagents and preparing the reaction, placing the cuvettes already has started the reaction. But still, we can identify the rate of the reaction.

The rate of the reaction is the slope of each graph that is considered the first and last time point values and measure it with respect to time. You will see that the slope of grey line is higher than that of others which says that rate of reaction of grey line is high.