Hi everyone! I'm a biochemistry student and I need some help for a homework, a laboratory report. The experiment consisted in following the thermal denaturation of the urease by fluorescence at 320 nm. Now, I need to find the value of the denatured fraction of the enzyme. I have the formula:
Fu = (Yf –Y) /(Yf –Yu)
where Fu = denatured fraction, Yf = expected fluorescence value for the folded portion, Yu = expected fluorescence value for the unfolded portion, and Y = measured fluorescence
For the expected fluorescence, I found some values in a scientific article that the professor referred to us:
S. Omar, M. Beauregard, Dissociation and unfolding of jack bean urease studied by fluorescence emission spectroscopy, Journal of Biotechnology, Volume 39, Issue 3, 1995, Pages 221-228, ISSN 0168-1656, https://doi.org/10.1016/0168-1656(95)00016-J
The found values are:
Yf = 500 000 photons/sec
Yu = 130 000 photons/sec
The mesured values (Y) are:
T(˚C) Y
46,9 344,1
50,6 299,9
55,6 254,5
58,8 210,3
64,5 192,4
65,5 144,8
66,6 147,0
67,6 89,1
68,6 92,3
69,3 89,5
70,5 60,9
71,0 38,0
71,9 4,5
72,8 -13,4
74,2 -8,5
75,2 -46,4
75,5 -56,4
77,8 -51,9
78,6 -127,5
80,7 -164,5
81,2 -177,5
83,4 -187,1
Unfortunately, the calculation of the denatured fraction does not work. The value of Fu is much too high, as you can see. What can I do?
Fu = (500 000 - 344,1)/(500 000 - 150 000)
Fu = 1,43!!!!!! Not possible!!!!!
Please help me!!! And sorry for my bad english!
You can't use published fluorescence intensity values. The reason is that fluorescence intensity measurements are relative, depending on many things. You have to actually make the all the measurements yourself. The formula you were given is telling you to calculate the proportion of the total measured fluorescence intensity difference between the native and fully denatured protein, using measurements you make yourself.
So let's use some arbitrary hypothetical intensity measurements. At the starting temperature, you measure a baseline of 100 RFU (relative fluorescence units). At the final temperature, at which the enzyme is fully denatured, you measure 1300 RFU. At some intermediate temperature, you measure 900 RFU. The fraction of denatured protein at the denatured temperature is (900 - 100)/(1300 - 100) = 0.67.
I agree with Adam B Shapiro about the importance of making your own measurements, but it is important that you also notice that in the equation you are working with two different units ¨photons pers second¨ (photon-counting method) for the Yf and Yu values and with a.u "arbitrary unit" (current-measuring instrument) for the Y values. I think that if you uniform the units, maybe you can get the expected values. For conversion from a.u to photons per second you can look in the instrument settings.
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