Chemical Forums
Specialty Chemistry Forums => Biochemistry and Chemical Biology Forum => Topic started by: 1101 on December 12, 2010, 04:53:03 PM

Ok so I'm supposed to be able to look at an enzyme's absorbance and determine specific activity from that but I really have no idea how. In this particular case I was given Chymotrypsin and pNitrophenyl Acetate. Supposedely chymotrypsin hydrolyzes pNitrophenyl Acetate (PNA) and that's the reaction I'm supposed to keep track of. During the lab we basically mixed Chymotrypsin, PNA, and a buffer and put a portion of this mixture in a spectrometer at given time intervals. The spectrometer analyzed the reaction for three minutes and gave us the slope. I assume by slope they meant absorbance per minute (though it could be per second). Anyways this is the data I got (note I've already turned the relevant assignment in, and gotten it wrong, I just want to understand this for the exam):
Time (Min)  Slope (Absorbance/Min?)
0  0.025
1  0.026
3  0.035
5  0.023
10  0.041
15  0.035
30  0.034
60  0.071
90  0.061
I am also given PNA's (the substrate) coefficient of extinction as 15000 M^1CM^1, and chymotrypsin's molecular weight of 23000 Da. Also the path length is 1cm.
Now I am entirely confused what to do with these values. I know the BeerLambert law says that
Absorbance = (Coefficient of extinction)*(path length)*(concentration).
So here's what I can plug in:
Absorbance = 15000(M^1)(cm^1)*1(cm^1)*(concentration in M)
Now I'm confused at multiple points here. Firstly they told us to record the slope of the reaction not the absorbance itself so how exactly do i fit this into the equation? Secondly I recorded multiple slopes am I supposed to average them or something or have i missed something? Thirdly I am supposed to find the concentration using this equation but wouldn't the units cancel out?

This reply may be too late for your assignment/test, but I thought I'd answer anyway.
First, I'm thinking those "slope" values you posted are actually raw absorbance values, from which you need to determine the slope. This slope will have units "change in absorbance per minute". You can calculate the slope by making a simple linear regression plot in Excel.
Your statement of Beer's law is correct, you can just rearrange it a bit. I'm going to use "dA" to mean "change in absorbance. dC is concentration, which you are trying to calculate.
dA/min = dc/min * pathlength * extinction coefficient
Rearrange : dc/min = dA/min* 1/pathlength * 1/extinction coefficient.
Plug in: dC/min = (slope you measured, units 1/min) *1 /(1cm) * 1/ (15,000 M^1 cm^1)
at this point the units of cm cancel (one appears in the top, one the bottom). And on the right side the units that remain are M per min. Or mol / (L*min).
You are left with dC/min = (slope you measured) / 15000. Again, units on both sides are M per min.
To calculate the specific activity, use the molecular weight you are give.
I hope this helps, others please feel free to add to or correct me!