i have been performing the aldose reducatse activity using commercially available purified rhAR enzyme from Sigma. following the standard protocol the change in the OD at 340nm is not very significant. i have queries regarding the reaction conditions and the preparation of the NADPH and DL-glyceraldehyde.
Under what temperature condition the assay should be performed?
In what buffer/solvent the NADPH and DL-glyceraldehyde should be prepared?
What should be the pH of the reaction mixture?
please provide your suggestions.
Hi there,
There is no particular trick in aldose reductase assay protocol. The reading of the following should address the whole bunch of questions:
https://ac.els-cdn.com/016748389500021L/1-s2.0-016748389500021L-main.pdf?_tid=10e6fc44-df4f-11e7-b65f-00000aab0f27&acdnat=1513091814_e7fe78ff5c72b408642863f667b0ebba
Here is another source of information containing human aldose reductase activity assay conditions:
Article The crystal structure of the aldose reductase·NADPH binary complex
NADPH solutions should be made fresh each day, kept on ice, then discarded.
I would need more details of the assay but I wonder whether it is just a technical problem. For solution any buffer would do (NADPH is soluble and glyceradehyde also; check anyway in any database, e.g Chemspider). For reaction pH I would just chech specifications of the product in the datasheet. Then one of the potential issues is that you are adding too much of the enzyme. Readings at 340 nm are high or low before and after adding the enzyme? Can it be that when you read at 340 nm the reaction has reached the end, i.e. readings are very low (and go slow) because there is little NADPH left?. The enzyme can also work with NADH, perhaps you may try both NADH and NADPH in parallel. And, finally: from my ignorance on this enzyme, I wonder why glyceraldehyde is used as substrate as it is not an aldose. I have gone to the "source" EC at IUPAC and the canonical reaction for the enzyme that I believe is "aldose reductase" (EC.1.1.1.21 isn't it?): Despite its official name as "aldehyde reductase" it is specified that the substrate for the the enzyme EC 1.1.1.21, is an aldose ( glyceraldehyde is not an aldose).
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