My experiment involve a dehydrogenase and I would like to identify the optimal substrate. For this an assay was established based on the use of DCPIP as cofactor, which when reduced the absorbance is lost (from blue to colorless) and this change can then be measured at 600 nm. The reaction (200 uL) contains 1 mM substrate, 0,1 mM DCPIP and 5 ug enzyme.
Then I tried to convert this information, from absorbance to specific activity as would it be for a publication. I would be grateful if the community check the attached Excel file for the operations I made and if this is the correct way to obtain specific activity values. As observed the values are very low, but on one substrate the result is clearly different. So I think you can not rule out catalytic activity, even if the values are very low, right?
Thank you for your observations!
Dear Luis:
The method described is good to follow dehydrogenase enzyme type. Your calculations as well as the plot look fine to me. Error bars could help to analyse much better the results. D-Lactate seems to be the best tested substrate.
Another methods to check dehydrogenase activities are based on the use of NADH or NADPH as coenzymes (to follow their oxidation by monitoring absorbance changes) or NAD+/ NADP+ (to follow their reduction by monitoring absorbance changes). The wavelengths useful to check the absorbance changes are usually 340 -380 nm. You will find many protocols related to these reactions in the literature.
Best regards,
Rosa María
In all cases, the absorbance changes are quite small and may therefore be unreliable. I agree with the suggestion of Rosa Martinez-Espinosa to use the simple assay format of measuring the decrease in absorbance at 340 nm of NADH or NADPH, if possible with your system, instead of DCPIP. The extinction coefficient change is 6.2 mM-1cm-1, if I remember correctly.
Thank you for your suggestions, I will try with direct NADH measurement.
Would you consider these raw values to be of a high absorbance and if using less DCPIP would increase sensitivity? Because while is widespread knowledge that values above 2 are unreliable due to the Beer law, I would guess this depends on the machine and the reagent. Other questions on ResearchGate suggest that values above 1 are unreliable. In the DCPIP case OD above 2 are not lineal anymore, but then there are two quantitative ranges, for low and high concentration.
Furthermore, machines seem not to take this into account. Our lab is considering purchasing a new plate reader, and Clariostar (BMG Labtech) dynamic range in the catalog is 0 - 4 OD. So my feeling if that by fixing on Beer law theoretical limits alone we would lose half of the information that can be acquired. Again, dpending on the analyte.
Amended on 28 January 2019
I must sadly and repeatedly say that the enzyme activity should be measured by HPLC-photometric method , which uniquely obeys to the Beer-Lambert law (please see file; J Chrom B Rat BIN LIP Km). Direct-utilization of photometer does not obey the Beer-Lambert law.
We have recently found that HPLC-photometric method gives three-fold higher results than the direct-utilization of photometer (please see Fig. 10 of the file; JCB Fucoidan Transport).
NAD and NADH may be separated by RP-HPLC, since lipoic acid/thioctic acid and reduced-form lipoic acid/dihydro-lipoic acid/dihydro-thioctic acid can be separable (please see file; Lipoyllysine determin).
I have searched for LDH in my protein database (please see file; HepG2 fucoidan).
Hepatoma HepG2 (cultured without edible Japanese Fucoidan) has L-Lactate dehydrogenase A-like 6A/LDHL2 at 2.5 μg/mg of cell protein.
LC tissue (with leprosy) has L-Lactate dehydrogenase A chain/LDH-M at 0.67 μg/mg of tissue protein.
Other two cancer tissues (HCC with PBC and HCC named No.6) have no LDH. Therefore, expression of LDH is not linked to cancer at all.
By the way, gene or protein expression is under the control of virus. Dehydrogenases linked to NADH is up-regulated by Corona virus and down-regulated by Human cytomegalovirus/HHV-5. It is interesting that human dehydrogenase with NADH is strictly controlled by these virus; i.e., human dehydrogenase with NADPH is controlled by another virus.
If your test shows that absorbance values are linear with respect to concentration throughout the range of interest, then there is no reason to restrict the OD range to 1 or 2. Some very good instruments are capable of linearly measuring higher absorbances, although the dynamic range may depend on the wavelength. If you are using a plate reader such as the Clariostar, you also have the option of reducing the sample volume, hence the optical path length, to reduce the absorbance of strongly absorbing signals. This is equivalent to using a thinner cuvette in a spectrophotometer.
If you have such high OD, that may be the reason, why you see only small decrease of absorbance. I'd recommend to dilute the DCPIP to half. That way you will get at least somewhere near 1.
Anyway, is the decrease proportional to the amount of added enzyme? If yes, it shall be OK. If no and you use crude extract, that doesn't have to mean anything, but if you use at least partly purified enzyme, that would suggest there is some problem.
Dear Luis
I agree with Tomáš Hluska about dilution to ensure the results near 1 and the suggestion to use NADH as co-factor with absoprtion at 340nm.
However, I have a doubt related to your data. What is the content of your Blank solution? There is a really high absorption to a blank... Data in 8-34 had been subtracted with the blank absorption (rows 35, 36 and 37)?
If the results in 8-34 have been previously substracted with blank absorption it´s OK... If not, you should do it before. Please take care with H54 cell.
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