What is Lowry method? Write its reaction with suitable example.

Introduction

The Lowry method is one of the most commonly used techniques for the quantitative estimation of proteins in a sample. Developed by Oliver H. Lowry in 1951, this method is highly sensitive and can detect protein concentrations as low as 5–10 micrograms per milliliter. It is widely used in biochemistry and molecular biology labs for protein quantification in various samples, including cell lysates, purified proteins, and enzyme preparations.

Principle of the Lowry Method

The Lowry method is based on two reactions:

1. Biuret Reaction: Proteins, under alkaline conditions, react with copper ions (Cu²⁺) to form a violet-colored complex. This is due to the peptide bonds in the protein that bind to copper ions.
2. Reduction of Folin-Ciocalteu Reagent: The copper-treated protein then reacts with the Folin-Ciocalteu reagent. This reagent contains phosphomolybdic and phosphotungstic acid, which get reduced by tyrosine and tryptophan residues in the protein. The reduced reagent forms a blue-colored complex whose intensity is directly proportional to the protein concentration.

Chemical Reaction Overview

• Step 1: Peptide bonds + Cu²⁺ (in alkaline condition) → Cu-protein complex (purple color)
• Step 2: Cu-protein complex + Folin reagent → Blue complex (absorbance measured)

Absorbance is measured at: 660–750 nm (usually at 750 nm)

Reagents Required

• Alkaline Copper Sulfate Reagent (Biuret reagent): Contains copper sulfate, sodium carbonate, and sodium potassium tartrate in an alkaline medium.
• Folin-Ciocalteu Reagent: A mixture of phosphomolybdic and phosphotungstic acids (usually diluted before use).
• Protein Sample: Unknown protein solution to be estimated.
• Standard Protein Solution: Bovine Serum Albumin (BSA) is commonly used as a reference.

Procedure Summary

1. Prepare a series of standard protein solutions using BSA in known concentrations (e.g., 20, 40, 60, 80, 100 µg/mL).
2. Add 1 mL of each standard or unknown protein solution to test tubes.
3. Add 5 mL of alkaline copper sulfate reagent to each tube and incubate for 10 minutes.
4. Add 0.5 mL of diluted Folin-Ciocalteu reagent and mix well.
5. Incubate for 30 minutes at room temperature or 37°C.
6. Measure the absorbance at 750 nm using a spectrophotometer.
7. Plot a standard curve and determine the protein concentration of unknown samples from the graph.

Example

If you want to estimate the protein content in a milk sample, you can treat the milk protein extract using the Lowry method and compare the absorbance with the BSA standard curve to find the concentration.

Advantages of the Lowry Method

• Highly sensitive (detects low levels of protein).
• Relatively simple and cost-effective.
• Good reproducibility and widely accepted.

Limitations

• Reagents are sensitive to light and time; timing is important.
• Certain substances (e.g., detergents, buffers) can interfere with the reaction.
• Color development varies with protein composition (tyrosine/tryptophan content).

Conclusion

The Lowry method is a reliable and widely used technique for protein estimation. It is based on colorimetric reactions involving copper ions and the Folin reagent. The resulting blue color intensity directly corresponds to protein concentration and is measured using a spectrophotometer. Though it requires careful handling of reagents, it remains a standard method in biochemical labs for protein quantification.