Recently, I tried to measure protease activity of different, crude venoms and also their fractions obtained after SEC. For this purpose I used Pierce™ Colorimetric Protease Assay Kit, which includes succinylated casein as a substrate, TNBSA as a dye and trypsin as a protease standard.
Unfortunately I encounter some problems because very often absorbance of my samples has negative values in relation to blanks. It is hard for me to explain what is the cause of such weird results. I have to underline that standard curve which was prepared based on manufacturer's reagents gave good results. To test whether it is not just analytical error I have also tested the kit to measure activity of pure PROMEGA trypsin (catalog number: V5111) and pure PROMEGA chymotrypsin (catalog number: V1061) and the absorbance of samples in relation to blanks was positive. I have also try different buffer (PBS, pH 7.4) and the results was comparable to those with Pierce Assay Buffer: positive for pure PROMEGA enzymes, positive for Agkistrodon contortix venom and negative for Naja ashei venom and its fractions. I do not know how it is possible to obtain negative absorbance. Whether there are some interactions between venom proteins and casein?
Maybe someone encountered similar problems while using this kit? Maybe I am making some sort of mistake?
Looking forward for any advices.
Hi there,
How negative are the OD values? Are these significant? Are these proportional to the venom amount introduced in the assay? Are you sure of the protease activity of the venom which gives these results? What is the composition of the venom solution? Do you set the zero with a sample having exactly the same composition?
Hi,
Thank you for your fast reply.
I will try to address to all of the questions:
1. How negative are the OD values? Are these significant? Are these proportional to the venom amount introduced in the assay?
Calculated A450 values (A450sample-A450blank) vary greatly between different samples. It could be as low as - 0.05 and also as high as - 0.800. I have not observed any corelation between concentration of venom and negative values of absorbance (so it was not proportional).
2. Are you sure of the protease activity of the venom which gives these results? What is the composition of the venom solution?
In general, we have tested four venoms from: Agkistrodon contortrix contortix, Naja ashei, Naja melanoleuca, Naja siamensis and also Naja ashei fractions obtained after SEC. Only the Calculated A450 values for Agkistrodon was positive, although there are lack of corelation between concentration and absorbance.
Our earlier results confirm the presence of proteases in all venoms. More specific:
Agkistrodon contortrix contortix (17% of serine proteases and 25% metalloproteinases)
Naja ashei (~2 % metalloproteinases)
Naja melanoleuca (14 % metalloproteinases)
Naja siamensis (~ 1% metalloproteinases)
3. Do you set the zero with a sample having exactly the same composition?
Final value of absorbance was calculated by subtracting absorbance of the blank from that of the sample.
Sample solution was prepared by adding 100 ul of Succinylated Casein Solution, 50 ul of sample (diluted venom or its fractions) and 50 ul TNBSA (after 20 min of incubation).
Blank contains Assay Buffer instead of substrate (casein) : 100 ul of Assay Buffer, 50 ul sample, 50 ul TNBSA
Every sample had its own blank.
Moreover, It is worth noting that measured venoms was diluted 250,500,750 and 1000 times. Lower dilutions than 250 gave very high OD values both for samples and blanks (even greater than 2.0).
I'd also like to add that whole test is based on the TNBSA reacting with primary amines which are generated after proteolytic cleavage of succinylated casein. It is possible that my venom samples contain some free amines which can react with TNBSA, however even that still not explain why the values of blanks would be higher than those from samples.
Interesting little mystery you have there. Some other suggestions to look at:
I start to worry about the accuracy of the spectrometer -- have you checked? One time I was having some similar weird issues with improper OD readings, and I discovered that a pencil had fallen into the optical path (must have fallen through a vent) -- removed that and things worked much better. Unless the control experiment that you described (purified protease) was PERFECT, you might think about carefully checking the spectrometer. Failing bulb or photomultiplier? With such high OD, I would expect you could at least see some of the color by eye, and get an idea of whether or not the readings in the spec are actually making sense.
Of course, the blank having high OD is a concern. Did you try a fresh sample of super-clean water as the blank -- maybe your assay buffer got contaminated? Maybe your cuvette is contaminated (again, speaking from experience)?
You might check the spectra also (not just single wavelength absorbance) as a way to check things. Or even by eye: do you get the same colors, or is the color changing? pH differences, other chemical contaminants, or even other enzymes could be altering the dye molecule, shifting the color/spectrum and giving you strange results.
Thank you for your advices.
Firstly, I am not sure if control test went perfect, however I obtained positive OD results for both enzymes in both dilutions. Twofold dilution of trypsin yielded in 1,26-fold decrease in OD (activity) in relation to not diluted enzyme. In the case of chymotrypsin, twofold dilution caused 2,34 times smaller OD value, so I think that results are not perfect but also not that bad. Moreover, every calibration curve prepared with trypsin from Kit were very good (R squared ~ 0.95).There are also other measurements that are conducted on this spectrophotometer and none of my colleagues complained about it.
Indeed, with very high OD sometimes I see differences in color by eye and it always agrees with further spectrophotometer measurements (Sometimes I actually see that my blank has clearly more intense yellow color than my sample).
I am trying to eliminate as many factors as I can so for example: each time I use new and sterile 96-well plates for every measurements. The buffer is delivered from manufacturer and is present both in sample and in blank.
In my case it looks like the only thing that is variable between sample and blank is the substrate (succinylated casein). It is possible that I am missing something but I think that maybe it is also possible that there are some interactions between substrate and the proteins in sample that surprisingly lead to lower OD results. However If that is the case maybe someone have idea how could I check that?
Again, thank you for your help!
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