Competitive/Non Competitive Inhibitors

**sara7000** · 12-01-2010, 03:39 PM

Hi,
I am doing my advanced higher biology project and am struggling to get my head around a few things. Would really appreciate if someone would help me.

I am investigating the effect of competitive (galactose) and non-competitive (Iodine) inhibitors on the activity of the enzyme B-Galactosidase. I am using ONPG instead of Lactose as a substrate.

Few questions:

1. Why does the initial absorbance reading for the diluted enzyme have to be between 0.3 and 0.5? Why this number? I thought it may be that the enzyme functions optimally at these readings, but then this idea contradicts my results as then my results should not go higher than this reading, which they do :O

2. Can I draw a graph of volume against absorbance reading? It shows a similar pattern to drawing concentration against absorbance but my ACTUAL INVESTIGATION, is the effect of increasing substrate concentration on com and non-com inhibition.

This is the link to the investigation procedures....It also mentions the 0.3-0.5 bit. The effect of competitive and non-competitive inhibitors on the enzyme ß-galactosidase

I really need help

Only two weeks to finish my write-up. Thanks

I am trying alot to find my own answers and have been avoiding asking on this forum but I couldn't think of anything else now lolz

**sara7000** · 13-01-2010, 04:02 AM

Anyone? Please

**AlaCourt** · 13-01-2010, 04:40 AM

Originally Posted by sara7000

Few questions:

1. Why does the initial absorbance reading for the diluted enzyme have to be between 0.3 and 0.5? Why this number? I thought it may be that the enzyme functions optimally at these readings, but then this idea contradicts my results as then my results should not go higher than this reading, which they do :O

2. Can I draw a graph of volume against absorbance reading? It shows a similar pattern to drawing concentration against absorbance but my ACTUAL INVESTIGATION, is the effect of increasing substrate concentration on com and non-com inhibition.

Right,
Question 1
They want the absorbance to be between 0.3 and 0.5, because that's when the colorimeter measures absorbance most accurately. I think once it gets above 1.0, it's loses a lot of its sensitivity, and above 2 it's pretty much saturated, so you really can't use readings up that high.

Question 2
Well, the volume shouldn't be changing, as substrate is soluble in solution, and so with the substrate being converted into products, these will (most probably) be soluble as well (and if they're not, I imagine their effects would be negligible). So only their concentrations should be changing, and NOT their volumes.
Btw, just so this is clear, you did keep the total volumes in the curvettes constant, right? Like, with increasing amounts of substrate, you took out the equivalent amount of water (or did you dilute the substrate appropriately before you added it to the reaction solution?).

Well, I hope that helps.

**sara7000** · 14-01-2010, 12:55 AM

Thak you that helps alot.
Yeah I kept the overall volumes the same by using a buffer. I was changing the volume of the substrate each time and I am not quite sure how that allows me to calculate the concentration? I drew a graph of volume of substrate against absorbance readings and got the same general shape as I would for a graph of concentration against abs readings. I asked my teacher if I could draw vol/absorbance reading and she said it was fine. But is volume not inversely proportional to concentration? I am cofused about how increasing the volume each time gives me a higher concentration?

I have tried to copy the table here:

Cuvette No ONPG Stock Solution Buffer ONPG X 20 Dilution
1 - 2.00 1.00
2 0.25 2.75 -
3 0.50 2.50 -
4 0.75 2.25 -
5 1.00 2.00 -

Thanks really appreciate the help

**sara7000** · 14-01-2010, 12:57 AM

The table for some reason isnt coming up properly but like for cuvette 1, it is no ONPG stock solution , 2ml of buffer and 1ml of x20 ONPG dilution added.

**AlaCourt** · 14-01-2010, 02:13 AM

Originally Posted by sara7000

Thak you that helps alot.
Yeah I kept the overall volumes the same by using a buffer. I was changing the volume of the substrate each time and I am not quite sure how that allows me to calculate the concentration? I drew a graph of volume of substrate against absorbance readings and got the same general shape as I would for a graph of concentration against abs readings. I asked my teacher if I could draw vol/absorbance reading and she said it was fine. But is volume not inversely proportional to concentration? I am cofused about how increasing the volume each time gives me a higher concentration?

To calculate the concentration of the substrate, you need to work out what fraction of the volume of the substrate added is in relation to the total volume of the solution. Then you multiply this fraction by the concentration of the stock solution of the substrate. That will give you the concentration.

For example, 5 ul (of substrate) divided by 50 ul (of total volume) = 0.1 (the fraction of the total volume).
0.1 multiplied by 5 mg/ml (concentration the stock solution) = 0.5 mg/ml is your concentration of substrate in solution.

You CAN use absorbance vs volume, its just that's not terribly scientific (you'd never see that in a science paper). It will yield similar results to absorbance vs concentration though.

It depends on what volume you're referring to. If you are adding a higher volume of SUBSTRATE [of a constant concentration], you're adding in more of the substrate, so you'd expect the concentration of substrate in the curvette to increase.
If you're referring to the volume of the curvette though (which you're keeping constant, so this SHOULDN'T be changing), an increased volume of the overall solution would mean that there would be relatively lower concentration in curvette, as you have the same amount of the substrate spread throughout more liquid.

Does that make sense?

**sara7000** · 14-01-2010, 02:41 AM

thank u soo much that makes everything very clear

And i think im more certain now of what I was doing till now. Thanks again for taking the time to write all of this. One last question, is cuvette 1 my control? or would i have to setup one separately with buffer and onpg only? I have already obtained a set of results for normal enYme activity by doing this already though? thnx again

Edit: I have discussed my control with my teacher and its all clear now. Thanks for all your help again