hydroxy proline detection

[rajeev]

Hello to all,

Is there any calorimetric assay that detects hydrolyzed protein (free amino acids including hydroxy proline) ?

I also understand, ninhydrin assay detect free amino acids (from hydrolyzed protein) except hydroxy proline, is that right?

Thanks in advance for your answers

RKB

[Mark Tracy]

There is a "colorimetric" reaction indeed with ninhydrin. Ninhydrin reacts with primary amino acids to give a strong purple color (570 nm) and with secondary amino acids to give a weak yellow color (440 nm). It is complicated by the fact that the purple product also absorbs at 440 nm, so you either need to separate the amino acids by chromatography or make some dodgy calculations. With ninhydrin, the chromatography must be before the reaction because the reaction removes the primary amine from the amino acid and you always get the same derivative.

[rajeev]

Hello,
Mark Tracy,

Thanks for your quick replay. Presently, i am trying to normalize the collagen level and collagen cross link level (HPLC detection) to the total protein from mouse FDL tendons.

Unfortunately i cannot measure the weight of the tendons for the normalization purpose. The only way to normalize (collagen and its cross links) is to have the total protein content from the tendons.

Because the tendons contain 60% collagen, i don't think i can depend on albumin assay (like Bradford method etc).

This is the reason, i thought if i can get the method to measure the hydrolyzed protein (all amino acids), that will give me the total protein level (with out missing hydroxy proline).

so, if there is any calculations require for the hydroxy proline measurement, i will take it.

Thanks for helping in this issue.
RKB

[Mark Tracy]

In this case, all you need is total elemental nitrogen. Send the tendon sample (or dried hydrolysate) out to a commercial lab that does elemental analysis. From the amino acid profile you know the theoretical %N of the collagen. It will be cheaper than doing the work yourself.