[nyan_nyan]

Hello!

I am currently developing an assay in which I can find out the hydroxyproline content of a lubricant I have made. However in the protocol I have to acid hydrolyse the lubricant and this causes burning and then caramelisation of the sugar in the lubricant leading to brown blobs forming in the solution. So I was thinking that I could filter this out but I am concerned that in the reaction by the sugars some of my hydroxyproline may have been involved so when I assay my clear lysate it wont tell me for sure how much hydroxyproline is in there.

The basis of my lubricant is pbs with 25% calf serum so I assume that the proteins and glycolipids etc in the serum are the source of the sugars because it doesn't happen in pbs alone.

I know this is a little vague but I just don't know where to start and any sort of perspective would be useful, do you think that the caramelisation reaction would involve any of the hydroxyproline or any other amino acids in the lubricant for some reason?

Thanks

[Arkcon]

Your digestion method is a little weird to me, and I'm wondering what your assay method is.  If I had a protein, and I wanted to assay its amino acid content, I would hydrolyze in HCl vapor, derivative the amino acids, and run a quick HPLC assay.  That wouldn't terribly attack sugars, and besides, they wouldn't derivitize, and so wouldn't show up on the chromatogram.  I really don't understand your terminology -- you have a lubricant, of PBS and BSA?  Do you mean something else?

[nyan_nyan]

Thankyou for your reply,

The assay I am modifying is one that has been developed a while ago and is quite established so that is why I have been trying to use it.  I am trying to adapt it from an assay that can assay the amount of hydroxyproline in a piece of tissue to one where I can assess it in a lubricant that contains tiny bits of tissue floating around in it.

I take the tissue, lyophilise it  and then hydrolyse it using HCL at 120 degrees celsius and this breaks the tissue down into amino acids. Then I use chloramine T to convert the hydroxyproline amino acids into pyrolles and then using Ehrlich's reagent you can make the pyrolles produce a colour that can be quantified with a plate reader. Because hydroxyproline makes up 1 of every 6 residues in a collagen II fibril I then times the value by 6 to find the total collagen content.

Normally this hydrolysing of tissue doesnt produce any brown stuff but when I do it on a lubricant which I have generated it goes brown because the lubricant contains calf serum and phosphate buffered saline  as well as a small amount of tissue that has broken off into the lubricant during testing. The brown product is basically from sugars caramelising. I dont mind if the sugars caramelise I just want to know if they will cause any of the hydroxyproline to be taken out of the hydrolysed solution. If they don't I can then continue my assay by just filtering the caramelised sugar out of the hydrolysed solution.

[discodermolide]

I would guess that some of your amino acid vanishes in the caramelization procedure.
Why not develop a quantitative reverse phase HPLC method, it won't require much substance and may be easier in the long run.