hydroxyproline assay - (Jun/25/2004 )

Hi there,
I'm presently doing an experiment looking at lung fibrosis and really need a protocol for an hydroxyproline content assay for lung tissue homogenates.
If anyone has one could they pass on the details?
Many thanks,
Will

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Solid material is precipitated from the tissue homogenate with 10% trichloroacetic acid by centrifugation at 5000g for 30minutes at 4°C. Samples are hydrolyzed at least 12 hours in 6 N HCl at 110°C.

Hydroxyproline content is quantified these days by Erlich’s reagent (Sigma Chemical Co. St. Louis, MO) and assayed by measuring the optical density at 555-562 nm. A hydroxyproline standard sample also from the same company is used to create a standard curve.
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This is the original reference for the protocol Sigma recommends. You might need an archaeologist to dig up this 43 year old paper-

Woessner JF, Jr: The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 1961, 93:440-447
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I have a good protocol and will try to unearth it from my home computer tonight. It is probably too long to post, so can I email the Word file to you?

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I am also highly interested in the hydroxyproline protocol and would be pleased to receive the word-file. jsellner@arcor.de

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I would be interested to have a copy of this method please.

martin.j.auger@aventis.com

The method we use is as follows and comes from UCSF:

Determination of Lung Hydroxyproline Content Lungs were analyzed for hydroxyproline content as previously described with slight modification.

Lungs were homogenized in 2.0 ml distilled water and incubated with 125 ul of 50% trichloroacetic acid on ice for 20 min. Samples were centrifuged and the pellet was mixed with 1 ml 12 N hydrochloric acid and baked at 110 °C for 14-18 h until samples were charred and dry. The samples were resuspended in 2 ml deionized water by incubating for 72 h at room temperature applying intermittent vortexing.

Serial dilutions of trans-4-hydroxy-L-proline standard (Sigma, Saint Louis, MO) were prepared starting at 0.5mg/ml.

200 ml of vortexed sample (or standard) was added to 500 ml 1.4% chloramine T in 0.5 M sodium acetate/10% isopropanol (Fisher Scientific, Pittsburgh, PA) and incubated for 20 min at room temperature.

Next, 500 ml of Ehrlich's solution (1.0 M p-dimethylaminobenzaldehyde in 70% isopropanol/30% perchloric acid) (Fisher Scientific, Pittsburgh, PA) was added, mixed, and incubated at 65 °C for 15 min.

After samples returned to room temperature, the optical density of each sample and standard was measured at 550 nm and the concentration of lung hydroxyproline was calculated from the hydroxyproline standard curve.

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