Enzymatic Hydroxyproline Assay Protocol

Introduction

This assay protocol is suitable for the colorimetric detection of hydroxyproline in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples using the Hydroxyproline Assay Kit (MAK008). hydroxyproline concentration is determined by the reaction of oxidized hydroxyproline with 4-(Dimethylamino)benzaldehyde (DMAB), which results in a colorimetric (560 nm) product, proportional to the hydroxyproline present. The linear range of detection for this assay is between 0.2–1.0 µg.

Reagents

• Oxidation Buffer (10 mL)
• Chloramine T Concentrate (0.6 mL)
• Perchloric Acid/Isopropanol Solution (5 mL)
• DMAB Concentrate in DMSO (5 mL)
• Hydroxyproline Standard, 1 mg/mL (0.1 mL)

Reagents and Equipment Required but Not Provided

• 96 well flat-bottom plate – it is recommended to use clear plates (Product No. M4436 or equivalent) for colorimetric assays.
• Spectrophotometric multiwell plate reader
• Concentrated (37% or ~12 M) Hydrochloric Acid (HCl, Product No. 320331 or equivalent)
• Activated charcoal (Product No. 242276 or equivalent)
• 120 °C Heating block
• Pipette compatible with concentrated HCl
• Centrifugal Evaporator or 60 °C oven
• Pressure-tight vial with PTFE-lined cap, or 2 mL polypropylene vial.

Preparation Instructions

• Briefly centrifuge vials before opening.
• Use ultrapure water for the preparation of reagents.
• To maintain reagent integrity, avoid repeated freeze/thaw cycles.
• Oxidation Buffer – allow buffer to come to room temperature before use.
• DMAB Concentrate – warm to room temperature to melt frozen solution just prior to use. Store protected from light and moisture at –20 °C.
  

Storage / Stability

The kit is shipped on wet ice. Storage at 2–8 °C, protected from light, is recommended.

Procedure

All samples and standards should be run in duplicate. Use ultrapure water for the preparation of standards and samples.

Hydroxyproline Standards for Colorimetric Detection

Dilute 10 µL of the 1 mg/mL Hydroxyproline Standard Solution with 90 µL of water to prepare a 0.1 mg/mL standard solution. Add 0, 2, 4, 6, 8, and 10 µL of the 0.1 mg/mL hydroxyproline standard solution into a 96 well plate, generating 0 (blank), 0.2, 0.4, 0.6, 0.8, and 1.0 µg/well standards.

Sample Preparation

To prepare serum or urine samples, transfer 100 µL of sample to a pressure-tight vial with PTFE-lined cap or 2.0 mL polypropylene tube. Add 100 µL of concentrated hydrochloric acid (HCl, ~12 M), cap tightly, and hydrolyze at 120 °C for 3 hours. Add 5 mg of activated charcoal, mix, and centrifuge at 13,000 x g for 2 minutes. Transfer 10–50 µL of supernatant to a 96 well plate.

Homogenize 10 mg tissue or cells in 100 µL of water and transfer to a pressure-tight vial with PTFE-lined cap or 2.0 mL polypropylene tube. Add 100 µL of concentrated hydrochloric acid (HCl, ~12 M), cap tightly, and hydrolyze at 120 °C for 3 hours. Transfer 10–50 µL of supernatant to a 96 well plate.

Evaporate all wells (samples and standards) to dryness under vacuum. Alternatively, place plates in a 60 °C oven to dry samples.

For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.

Assay Reaction

Preparation of Assay Reagents - the following 2 assay reagents are stable for 2–3 hours after preparation, and should be prepared after sample preparation, just prior to the start of the assay.

Chloramine T/Oxidation Buffer Mixture – 100 µL is required for each reaction well. For each well, add 6 µL of Chloramine T Concentrate to 94 µL of Oxidation Buffer and mix well.

Diluted DMAB Reagent – 100 µL is required for each reaction well. For each well, add 50 µL of DMAB Concentrate to 50 µL of Perchloric Acid/Isopropanol Solution and mix well.

1. Add 100 µL of the Chloramine T/Oxidation Buffer Mixture to each sample and standard well. Incubate at room temperature for 5 minutes.
2. Add 100 µL of the Diluted DMAB Reagent to each sample and standard well, and incubate for 90 minutes at 60 °C.
3. Measure the absorbance at 560 nm (A₅₆₀).

Results

Calculations

The background for the assay is the value obtained for the 0 (blank) hydroxyproline standard. Correct for the background by subtracting the blank value from all readings. Background values can be significant and must be subtracted from all readings.

Use the values obtained from the appropriate hydroxyproline standards to plot a standard curve. The amount of hydroxyproline present in the samples may be determined from the standard curve.

Note: A new standard curve must be set up each time the assay is run.

Concentration of Hydroxyproline

Sa/Sv = C
Sa = Amount of hydroxyproline in unknown sample (µg) from standard curve
Sv = Sample volume (µL) added into the wells
C = Concentration of hydroxyproline in sample

Sample Calculation

Amount of hydroxyproline (Sa) = 5.84 µg
Sample volume (Sv) = 50 µL

Concentration of hydroxyproline in sample
5.84 µg/50 µL = 0.1168 µg/µL