My plant biomass contain a secondary metabolite indican, that get hydrolysed when I soak the plant biomass in water.
Now there is two product that I can get from this hydrolysis.
1. glucose
2. An unstable compound (indoxyl)
As I can not measure indoxyl, to know how much indican is hydrolysed I have to measure the glucose that is liberated out from the cell to the water medium that was used for hydrolysis.
Does glucose from plant biomass can liberat from other source too that will interfere in accurate measurement of my target glucose molecule?
Glucose will come from cellulose andhemicellulose, but if you are just soaking your biomass in water (no acids), then it is imposssible that glucose will come from cellulose or hemi. So my guess whatever glucose you are getting is from hydrolysis of indican.
Dear Shrikant!
Can glucose come from leaf in alkaline condition too?
Can you describe it more elaborately?
If you are dealing with leafy biomass, which in my opinion do not contain cellulose and hemicellulose. You follow NREL protocol and find out the sugar (glucose, xylose, arabinose) content. Then you can co-relate the glucose with the Indican content. If its more then you have some other source of gluose coming out from leaf. If not then you can say that glucose released is from indcan hyrolysis only.
Alkaline condition depending on the experimental conditions can give glucose.
What about bubbling with oxygen to oxidize indoxil to indigo and then measuring indigo? (The idea adapted from http://2013.igem.org/Team:Berkeley/Project/FMO). Seem much cleaner then trying to estimate where does the glucose come from...
Yoram
Thank you Gerchman for your suggestion.
I have already adapted that .I want some direct measurement method after hydrolysis.
There are so many highly complex metabolites in the average leave that you will run into all sorts of trouble approximating to the sugar equivalents. Your best bet is to do a solvent based method in a non-hydrolysing environment e.g. dry dichloromethane. If your compound is generally unstable best to add an inert atmosphere too (nitrogen or argon). Do it all with the freshest material you can find and keep it as cold as possible to protect your compound. Crushing the leaves up with a mortar and pestle with liquid nitrogen is a good method.
If you're looking for glucose which is a metabolite, for sure that any monomer(glucose) arising from cellulose/hemicellulose decomposition will be released by only soaking any part of the plant in pure water. There are lots of techniques for measuring glucose. And you can extract it with a organic polar solvent (i.e. THF) for further evaporation and concentration. Then you just have to apply one of the plenty reducing sugars quantification methods that are already extensively reported. There are even some kits available. But of course this is having as principle that glucose will not result as a metabolite from any other product present.
I agree with Mushafau. Depending on the part of the plant that you have, you can have a significant amount of free sugars that can dissolve in the water when you soak the material. I believe it is very likely that you are going to have interferences in that measurement that you are proposing.
Depending on the anatomical fraction it is possible to release free glucose, sucrose (which can easily hydrolyze to glucose and fructose) or from labile parts of starch into a water extraction. It would be very unlikely to release glucose from the structural polymers (cellulose or hemicellulose). It would seem like there would be a considerable chance for glucose interferences in this proposed assay.
Thank you all For your suggestions !
Mr. Oke!
I am working on leaves of Indigofera tinctoria, which is coming under legume family
Dear Lopa,
indican is a bets-glucoside. A simple experiment I would do would be beta-glucosidase assay to get any preluminary quantification of indican. A pNPG (beta pNPG) assay should be able to get you there. It is a simple experiment which you can do in a short amount of time (with no need to soak your plant in water).
I am sorry that due to posting this from mobile, I am unable to provide you a reference paper at the moment.
I want to measure the indcan of biomass,as I am using my biomass for my experimentation
Dear Lopa,
I hope that these papers will be helpful to your experiment.
Regards,
Avinash
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs