Chem 330 L
Determining protein content in liquid protein drinks
and protein powders using the biuret reagent
Introduction
The biuret assay is a method that can be used to determine the amount of protein in various
samples. In this assay, copper (II) ions interact with four nitrogen atoms in two peptide bonds.
A purple colored complex is formed as a result of this interactions and is proportional to the
amount of protein present in the assay. The biuret assay is one of many assays used for
determining protein content. It can be used to determine protein in the range from one to ten mg
protein per ml solution. The assay is a relatively simple spectrophotometric assay that measures
the absorbance of the purple complex at 540 nm. Protein content is determined by extrapolating
the amount of protein from a standard curve of absorbance vs the amount of protein in the assay.
Normally, bovine serum albumin (BSA) is used as a standard although a purified whey protein
fraction could also be used.
There are several potential problems in measuring the protein content because of interfering
substances. We have found that some of these substances include food dyes (red dye # 40),
some types of vitamins (B12), and certain salts (CaCl2). Another problem is related to what type
of protein is used as a standard for generating a standard curve.
We are going to measure the protein content in a liquid protein drink containing whey protein
Kellog’s K2O protein water
Experimental
1. Take 1 mL of the K2O water and add 0.2 mL of 50% trichloroacetic acid (TCA) in a
microfuge tube (tube a). Repeat this same procedure for another sample (tube b). Mix well and
let the sample stay on ice for 5 min. Then centrifuge the sample at 10,000 rpm for 5 min.
Remove the supernatant and add 1 ml of acetone to the microfuge tube a. Mix well and
centrifuge for 2 min at 10,000 for 3 min. Remove the supernatant. Add 1 mL of 1 M NaOH to
the tube and dissolve the protein. To tube b, add 1 mL of 1 M NaOH to dissolve he protein.
Prepare a series of tubes containing BSA or whey protein as a standard. Also prepare samples of
K2O that was not precipitated with TCA and your TCA precipitated samples. (see below) Do all
samples and standards in duplicates.
2. Preparation of standards and samples for the biuret assay
Test Tube
1
2
3
4
5
6
BSA/whey protein
mL
0
0.2
0.4
0.6
0.8
1.0
7
K2O TCA ppt (tube a)
0.50
0.50
3
K2O TCA ppt (tube b)
0.5
0.5
3
K2O original sample
0.50
0.50
3
8
9
BSA/whey protein
mg
0
2
4
6
8
10
water
mL
1
0.8
0.6
0.4
0.2
0
biuret
mL
3
3
3
3
3
3
Abs
Abs
Using your standards, plot the average absorbance vs the amount of protein in the test tube (mg).
You should get a relatively straight line. Take your absorbencies from your K2O samples and
extrapolate the corresponding protein content. Divide the protein by the volume of sample used
to find the concentration in mg/mL and to take into account the original K2o sample volume.
Determine the protein content in the drink from the label listing. Compare your estimate of the
protein content with regard to the original sample, the TCA precipitated sample in tube a, and the
TCA precipitated sample in tube b. If one of the groups used whey protein as a standard,
compare your results to their results.