Hydroxyproline Assay Using NaBr/NaOCl

Materials and Reagents

1. NaOCl (Lab bleach)
   
2. NaOH
   
3. NaBr
   
4. 6 N HCl
   
5. Dilute hypobromite
   
6. p.dimethylaminobenzaldehyde (Sigma-Aldrich, catalog number: 156477 )
   
7. n.propanol (Sigma-Aldrich, catalog number: 402893 )
   

Equipment

1. 2 ml screw-cap microtube (SARSTEDT AG)
   
2. Microplate reader or spectrophotometer
   

Procedure

1. Prepare dilute sodium hypobromite (NaOBr) from NaOCl, mix equal volumes (a) + (b) (e.g. 5 ml each) (prepare fresh weekly-store at 4 °C).
   1. Add 775 μl lab bleach to 10 ml 4% NaOH (fresh weekly).
      
   2. Prepare 100 mM NaBr (1.03 g in 100 ml 4% NaOH) (stable).
      
2. Add 250 μl aqueous sample to 2 ml screw-cap microtubes (e.g. SARSTEDT AG).
   
3. Add 500 μl dilute hypobromite to:
   
   1. Analysis samples each in 250 μl distilled water.
      
   2. Hyp standards of 2.5, 5.0, 7.5 and 10 μg, each in 250 μl distilled water.
      
   3. A reagent blank contains reagents plus an additional 250 μl distilled water.
      
4. Mix and leave for 5 min to oxidize at room temperature.
   
5. Add 250 μl 6 N HCl.
   
6. Add 500 μl 5% p.dimethylaminobenzaldehyde in n.propanol (total volume = 1.5 ml).
   
7. Mix and heat at 70 °C for 15 min, then cool in ice-water.
   
8. Measure absorbancy of samples and standards at 560 nm against the reagent blank. e.g. 10 μg Hyp → ~680 mAUs
   
9. Construct the standard curve and calculate sample values by interpolation.
   

Recipes

1. This assay determines free hydroxyproline, best prepared by hydrolysis peptide bonds in the sample to be analysed (6 N HCl at 110 °C for 18 h) followed by removal of HCl in vacuo and redissolving the hydrolysate in distilled water. Interestingly free O-glycosylated Hyp can be assayed directly.
2. Use aqueous Hyp standards over a 2.5 to 10 μg range. It is convenient to prepare a Hyp standard of 100 μg/ml in distilled water stored frozen; mix well after thawing! The reagent blank contains all the reagents with distilled water as a substitute for the sample.
   
3. 5% w/w p.dimethylaminobenzaldehyde in n-propanol.
   

Acknowledgments

This protocol is adapted from Kivirikko and Liesmaa (1959) and Lamport et al. (2012).

References

1. Kivirikko, K. I. and Liesmaa, M. A. (1959). A colorimetric method for determination of hydroxyproline in tissue hydrolysates. Scandinavian J Clin Lab 11(2): 128-133.
   
2. Lamport, D. T., Kieliszewski, M. J., Chen, Y. and Cannon, M. C. (2011). Role of the extensin superfamily in primary cell wall architecture. Plant Physiol 156(1): 11-19.
   
3. Vogel, A. I. (1961). A text-book of quantitative inorganic analysis. 1-1216, Third Edition. Publisher: Longmans.