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Topic: Mechanism of Hydroxyproline Assay  (Read 7858 times)

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Offline sparkleys

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Mechanism of Hydroxyproline Assay
« on: July 23, 2012, 09:59:35 PM »
Hello, I'm rusty on my organic chemistry but am trying to find a mechanism for a particular assay we've been running to isolate hydroxyproline content. The assay we are using comes from Woessner: http://www.aufsi.auburn.edu/recommendedmethods/05A02h.pdf

The original assay they reference is in german which unfortunately I can't access/read.

Sample: Muscle dissolved for 20 hrs in 6N HCL. After, drop of methyl red indicator is added and 6N NaOH is titrated until pH is 6-7.

Method (Mixtures are described below)
1. Chloramine-T Mixture is added to Sample, mixed, and let stand for 20 minutes at room temperature.
2. 3.15M Perchloric Acid is added, mixed, and let stand for 5 minutes at room temperature.
3. p-dimethylaminobenzaldehyde Mixture is added, mixed, and heated at 60C for 20 minutes.
4. Benzene extraction (Benzene is added, mixed, discarded)
5. Read at 557um within 10 minutes
6. 30% H2O2 is added, mixed, and read again at 557um within 5 minutes.

Chloramine-T Mixture
1.41g Chloramine T dissolved in 20mL water. 30mL of methyl cellusolve and 50mL buffer added.

p-dimethylaminobenzaldehyde Mixture (20% Mixture)
20g p-dimethylaminobenzaldehyde is added to give final volume of 100mL. Heat until dissolved.

I believe methyl cellusolve is an organic solvent used. I'm not sure on its purpose. The original assay states, "Method II extends the Stegemann method by benzene extraction and peroxide treatment, thus permitting the determination of hydroxyproline in samples containing large proportions of other amino acids." I have no idea how it does this.

Any thoughts/help is appreciated.

Offline sparkleys

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Re: Mechanism of Hydroxyproline Assay
« Reply #1 on: July 24, 2012, 07:28:13 PM »

These are the structures and the mechanism I'm looking for.

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