[Maciejczyk]

Hi, I need to write up the experiment, and I am struggling a bit with the calculations.

I have got 3g/100 mL suspension of yeast in 20mM phosphate buffer pH 7.5.

I made up solution of total volume 4mL:

- 2.9 mL potassium phosphte buffer ph 7.5
- 1 mL of yeast suspension
- 0.1 mL 50 mM d-glucose

I had to measure the rate of oxygen consumption by noting the oxygen concentration every 15 sec. over a 4 min period, that is my data:

Time (sec)  D-glucose
15       95
30       92
45       89
60       89
75       85
90       84
105       82
120       81
135       80
150       79
165       78
180       77
195       76
210       75
225       74
240       73


Now I have 2 questions to answer:

1. Calculate the rate of oxygen uptake in a umol/min/g yeast suspension, assume oxygen solubility of 0.25 umol/mL.

I have done it this way:

95-73 = (22 / 240)*60 = 5.5% O2/min
5.5% * 1umol O2 = 0.055 umol/min
0.055 umol / min / 30mg = 0.0018 umol/min/mg

Do you think it is correct?

2. Calculate the rate of sugar uptake in umol/g yeast/min for d-glucose.

Now this is the one that I struggle with.

I have measured the absorbance of d-glucose at 520nm (same solution as above):

0' - 1.191
30' - 0.524

Now I cannot use Beer-Lambert law beacuse I can't use extinction coefficient to to use of DNSA.

Could anyone help? I am stuck.

[Borek]

Time (sec)  D-glucose

Glucose or oxygen?

Now I cannot use Beer-Lambert law beacuse I can't use extinction coefficient to to use of DNSA.

Please elaborate, I don't get what you mean.

[Maciejczyk]

It is oxygen uptake for soultion with d-glucose.

What I meant is that I would use Beer-Lambert law, and find extinction coefficient to calculate the concentration. But I can not do that because of presence of 1ml of DNSA which reacts with reducing sugars and absorbs much light at 540nm.


Sorry for my clumsy writing.

[Borek]

Then what was the absorbance measured for?

[Arkcon]

I have measured the absorbance of d-glucose at 520nm (same solution as above):

0' - 1.191
30' - 0.524

Now I cannot use Beer-Lambert law beacuse I can't use extinction coefficient to to use of DNSA.

Could anyone help? I am stuck.

I suppose we can infer you have some sort of assay for dissolved oxygen that uses some chromaphore you measure with a spectrophotometer.  Since you know your observance result, and you initial concentration (as Borek: said not clear to us weather you measure loss of glucose or loss of oxygen) of something, can't you compute the extinction coefficient, and use that value for unknown steps?

[Maciejczyk]

I have to compare D-glucose with substrates for aerobic respiration by yeast.
The ability of yeast to oxidise these substrate was measured using an oxygen electrode.

If the yeast are unable to oxidise any of these substrates it may be because:
a)The yeast is unable to take up the substrate from the medium, or,
b)The yeast cannot metabolise the substrate once it has been taken up.

To distinguish between these alternatives I have to measure the rate of uptake of the sugar.

Sugar uptake would be measured by measuring the decrease in sugar concentration in the medium over a period of time. D-glucose is a reducing sugar and can be measured by the DNSA reaction.

Now I have to calculate the rate of sugar uptake in umol/g yeast/min.