I checked the previous answers :)
So my enzyme kinetic assay is based on DCPIP absorbance at 3 mM, which is outside of the linear optical range to measure continuously on a plate reader. Then I have to stop the reaction but the common method, pH change with acid doesn't work because pH changes alter the absorbance of DCPIP. Ethanol and other alcohols do the same. I tried urea 8 M but it seems that this is not denaturing the protein, or that is still active (3 measurements within 5 minutes difference gave different activity values). I'm trying to make a higher urea concentration to see if that works, but other than that and freezing with liquid nitrogen what else can I try?
You can incubate your sample -80degree C for 2min. Most of enzymatic reaction stops at freezing temp.
Regards
Abhishek
The simplest change to make is to reduce the volume of the reaction. This will shorten the pathlength and thereby reduce the absorbance proportionately.
Amended on 9 May 2018
I sadly and repeatedly am saying that the direct utilization of photometer without purification such as Plate Reader is not quantitative due to violence against Lambert-Beer's law. I have recently demonstrated that HPLC-photometric value is c.a. 2-fold higher than the value obtained by direct-use of photometer (please see Fig. 10 of the file; JCB Fucoidan Transport).
HPLC-enzyme assay is uniquely quantitative (please see file; J Chrom B rat BIN LIP Km). The reliable stopping method for biotinidase and lipoamidase has been described in this file. As shown in Table 1, addition of 0.05 mL Methanol–Acetonitrile (1:1, v/v) solution (stopping solution) to 0.10 mL of the reaction mixture has been found to be successful. Interestingly, ethanol and ethyl ether can not stop the reaction. The reason of this phenomenon has not yet been clearly clarified, however this methanol-acetonitrile stopping method is only applicable to HPLC method. Direct photometry can not be used, since this stopping solution induces protein aggegation and turbidness. On the other hand, only centrifugation of this turbid solution (stopping solution + reaction mixture; 0.15 mL) or filtration is suitable to inject into the HPLC system.
Nowadays, leasing company of HPLC-system is present in Tokyo and also may be everywhere including Germany.
By the way, I have previously surprized that oxidized-form lipoic acid and reduced-form lipoic acid (relative difference of molecular mass is only 2) are completely separated by RP-HPLC (please see file; dihydro LA).
Then, you may safely separate oxidized- and reduced-form of 2,6-Dichlorophenolindophenol (DCPIP, DCIP or DPIP) by RP-HPLC.
Furthermore, I like the answer of Dr. Tapan Kumar Mazumder; i.e., only separation between the enzyme and the substrate is sufficient to stop the reaction. Thus, injection into HPLC system is sufficient to stop the reaction. Therefore, use of several HPLC machines may be required to measure the initial-rate determinations.
Then, I hope you your success by using HPLC, since "Slow and (but) steady wins the race".
I think what Adam is suggesting is to find an optical cell with a short enough light path to do your assay continuously. You should be able to find 5 mm cuvettes pretty easily, 2mm if you really try. It may also help to monitor at a wavelength somewhat off of the maximum, say 550 or 570 instead of 600 nm.
If absorbance measurement requires diluting by a significant factor, you might try pipetting your timepoint aliquots into an alcohol (methanol, ethanol, 2-propanol). To see if a particular alcohol works, transfer an exzyme sample, mix and centrifuge, then measure absorbance, let stand a few minutes and measure again. If the abs isn't stable, try the next alcohol. Disposable cuvettes for this should probably be made of polymethacrylate - polystyrene surfaces may cloud because of the solvent. This and the off-maximum method both require you to do new standardizations rather than just using the literature molar absorbance to calculate oxidized DCPIP conc.
If you can't make these work, you can also heat-denature protein from your timepoint aliquots. Use glass vials or tubes in a "Dry Bath" aluminum heating block that's been set at sufficiently high temp to ensure the enzyme denatures immediately. 95 oC is pretty sure to work for enzymes from non-thermophiles; trial-and-error can give you a usable lower denaturation temp. The 1 mL flat bottom tubes with plastic stoppers they sell for HPLC autosamplers should work well & Al blocks with holes for these tubes (~ 7-8 mm dia) are widely available. Allow appropriate volumes of diluent buffer in these stoppered tubes a few minutes to come to temperature, then pipette enzyme reaction aliquots at appropriate times and cap. Remove quenched samples from the heat as soon as you are confident that the enzyme reaction will not resume. Handling times should be as uniform as you can make them. Dilute as needed and spin the samples down in a microcentrifuge before transferring a suitable volume for absorbance measurement.
You can incubate your sample -80degree C for 2min. Most of enzymatic reaction stops at freezing temp.
Regards
Abhishek
Subject the reaction mixture to heat which can denature the protein. Since heat denaturation is reversible the time gap between heating and absorbance measurement must be less.
Denaturation is a process.
Other than that exposure to UV rays, beta, gamma and x -rays also inactivated some enzymes due to formation of peroxidases.
Have you tried other organic solvents such as acetonitrile? ACN has a lower absorbance compared to methanol
As far as I guess, your purpose is not to denature the proctein but to stop the reaction for a while and then reactivate it to continue your measurement, you may need mild conditions, so, try to place the your working reaction mixture at temperatures outside your working temperature range of the enzyme, such as dipping in cold water or in ice etc, rather than adding something that may change pH and or enzyme/substrate concns, reaction volume etc. Good luck
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