With nothing but water in the cuvette, a rising far-UV absorbance with time suggests to me that the quartz cuvette you are using is not clean enough. Make sure it is absolutely clean for each new measurement, because any organic residues will become crosslinked by the UV light exposure, causing an increase in absorbance.

Once the substrate and enzyme are mixed thoroughly at the start of the reaction, it should not be necessary to keep mixing them, as long as there are no particulates that settle out if not stirred.

Watch out for the growth of bubbles on the walls of the cuvette during prolonged incubations, which will cause erratic absorbance values.

Hi Adam,

Well, if it really isn't necessary to keep mixing the reaction, my problems would be solved, sinced I've noted that if I don't mix the water, the absorbance keeps still. I've tried cleaning the cuvette with 2M HCl (as I've read) and a lot of water, but it seems that it hasn't been enough.

How can I be sure that mixing isn't necessary? I say that because, for plates, the equipment that I use for the readings offers the option of 'shaking' before each reading, when I'm doing a kinect assay.

Thanks

hi guilherme

i have experience using spectrophotometer in 2 years ago, maybe you can make sure your cuvette first or using the new cuvette Quartz. then, i using the ethanol or water be a blank. then in menu, always using blank correction.

hope can answer

Shaking should not be necessary before each reading for a homogeneous solution, but should be used for a suspension in which the particles tend to settle. The solution should be mixed at the start, which can be done using the shaking function of the plate reader or with a separate plate shaker.

There may be exceptional cases when continuous mixing of homogeneous solutions is needed. One example would be where the reaction consumes oxygen and you want to replenish the oxygen from the air.

I see...

I thought mixing was necessary so the enzyme could access new molecules of substrate, but it seems that I was wrong.

Thanks for the answers, Adam

You can test this idea by performing the same measurement, either mixing before each measurement or not, and comparing the reaction rates and the length of time during which the reaction rate is constant.

That would be great, if I wasn't suspecting that my cuvette is releasing something when I mix the reaction by turning it upside down.

I guess that my best bet, by now, would be preparing a higher reaction volume, and keep taking 500uL from this volume and measuring, then discarding, since I've observed that, when mixing the reaction in an eppendorf tube, the absorption doesn't rise unexpectedly. Then, I could compare this to a reaction only initially mixed.

There is a problem that has not been sorted out if the absorbance of pure water in a quartz cell keeps increasing.  What happens with an empty cell and in the absence of a cell?    How big is the change?

So, Paul, an unmixed cuvette gives always the same reading, so my best bet still is that there is some kind of contamination. An empty cell and no cell at all gives fluctuating values, what I suppose is expected.

When there is only water, if I keep mixing and reading, after 5 minutes I'll have observed an increasing around 20 mAU.

Thanks for the tip, Ivan. I'm about to test what I described before, and if it still goes unsuccessful, I'll go after this Hellmanex III.

I like to use chromic acid for cleaning quartz cuvettes. It works very well, it doesn''t take long (~30 minutes), it doesn't harm the cuvettes, and the solution can be reused many times so it generates very little chemical waste. Care must be taken, of course, because it is highly corrosive. The cuvette should be dry when the solution is added to avoid localized heating, and the cuvette must be very thoroughly washed with distilled water afterwards to remove any chromic acid residue. It's a good idea to have at least 2 cuvettes so that you can wash one while the other is in use.

I prefer soaking the cells over night in 5% peroxyacetic acid. If you still have trouble prepare the solution with warmed up water. 

For intermediate short term cleaning: Always immediately after measuring flush the cuvette first with a lot of water and then with ethanol/isopropanol/... turn it and put it onto a soaking leaf for drying completely.

Chromic acid works excellent, however is a mess, because you have always lurking carcinogenic Cr-VI around and chromylchloride, the latter,  if the used solution gets contaminated with Chloride.

Variation/ fluctuation in readings, even with pure water in the cuvette,  can be because of a unclean cuvette. There could be some residues sticking to the cuvette which are invisible to the naked eye. It is advisable to clean the cuvette, preferable using Chromic acid, After the chromic acid treatment, make sure that the cuvette is rinsed well with plenty of distilled water and then air dried. 

"An empty cell and no cell at all gives fluctuating values, what I suppose is expected." You should get a certain value, which is lower than the measured with cell. Based on what you observed, I doubt the problem can be your UV-vis detector rather than the cell. Why not test the same sample using a UV-vis detector in other labs.