CONCEPCION, Elaine F.

Chem 28 HW #3 #4

A. How does BIURET differ from KJELDAHL?

What is determined
BIURET
Total protein concentration
Colorimetric method
KJELDAHL
Total nitrogen content and
protein (multiplied by
conversion factor)

Reference/s:

BIURET assay method

- Quantitatively determines total protein concentration[1]

- Colorimetric method (simple and rapid)[1]
- Principle: The –CO-NH- bond (peptide) in polypeptide chain reacts with copper sulfate in an
alkaline medium to give a purple color which can be measured at 540 nm[2][5]
- Involves a standard/ calibration curve to calculate the concentration of protein in a given
sample[2]
- Biuret reagent: potassium sodium tartrate + cupric sulfate pentahydrate + NaOH + potassium
iodide; diluted with d. H2O[3]
- Has a sensitivity of 0-1 mg[4]
- Has high accuracy, with no dependence on amino acid composition[4]
- Has interference with amino-group [(NH4)2SO4][4]
- Principle: Reaction of Cu2+ with peptide bonds to form blue adduct[5]
- Most common technique for measuring total protein using automated chemistry analyzers[5]
- Does not distinguish between proteins because the reaction is specific for peptide bonds, which
are common to all proteins. Therefore, the method is used to quantify total protein content of
serum.[5]

[1] Wrolstad, R. E.; Acree, T. E.; Decker, E. A.; Penner, M. H.; Reid, D. S.; Schwartz, S. J.; Shoemaker, C. F.;
Smith, D.; Sporns, P. Handbook of Food Analytical Chemistry: Water, Proteins, Enzymes, Lipids, and
Carbohydrates; Wiley: USA, 2000-2005; pp 87.

[2] Mahesha, H. B. Estimation of Protein by Biuret Method. 2012.

[3] Savory, J.; Pu, P. H.; Sunderman, Jr., F. W. A Biuret Method for Determination of Protein in Normal
Urine. Clin. Chem. 1968, 14, 1162.

[4] Martina, V.; Vojtech, K. A Comparison of Biuret, Lowry and Bradford Methods for Measuring the
Egg’s Proteins. [online] p 395
[5] Bertholf, R. L. Proteins and Albumin. Lab Medicine. 2014, 45, 28 [online]

 Check references: (a) Copeland R. A. 1994. Methods for Protein Quantitation. Methods for
Protein Analysis [online]. Available from: http://link.springer.com/chapter/10.1007/978-1-4757-
1505-7_3 (b) Malim T., Ridzuan P. D. 2010. Principle in Biochemistry. [online]. Available from:
http://www.slideshare.net/UmiBiee /lowry-vs-biuret-final-1 (c)

KJELDAHL method (981.10 AOAC)

- Determines total nitrogen content and protein as the nitrogen content of the sample multiplied
by a conversion factor[1]
- Sample is digested in sulfuric acid, using CuSO4/TiO2 as catalysts, converting N to NH3, which is
distilled and titrated[1]
- Applicable to a wide range of organic matter including raw materials, ingredients, and finished
products from animals, cereals, and oilseeds[1]
- Principle: Acid digestion, measurement of liberated ammonia by titration with Nessier reagent,
multiply nitrogen by 6.25[5]
- Indirect protein determination method; nitrogen is released into a strong acid and the content is
measured after neutralization and titration[6]

[1] Wrolstad, R. E.; Acree, T. E.; Decker, E. A.; Penner, M. H.; Reid, D. S.; Schwartz, S. J.; Shoemaker, C. F.;
Smith, D.; Sporns, P. Handbook of Food Analytical Chemistry: Water, Proteins, Enzymes, Lipids, and
Carbohydrates; Wiley: USA, 2000-2005; pp 105.

[6] Mæhre, H. K.; Dalheim, L.; Edvinsen, G. K.; Elvevoll, E. O.; Jensen, I. Protein Determination – Method
Matters. Foods, 2018, 7, 4.

B. What are the ADVANTAGES and DISADVANTAGES of BIURET METHOD?

ADVANTAGES
Simple
Rapid color development time; constant
color intensity for at least 30 minutes
Sensitive to low concentrations of proteins Lack of sensitivity
High accuracy, not dependent on amino acid
composition
DISADVANTAGES
Time consuming (due to the amount of
processes a food sample must undergo
before it can be analyzed)
Sometimes difficult to quantitatively extract
proteins from certain processed foods
(proteins become aggregated or covalently
bound with other substances)

Reference/s:
 [] University of Massachusetts Amherst. Analysis of Proteins.
https://people.umass.edu/~mcclemen/581Proteins.html (accessed September 10, 2019)
[] Ohio Northern Univeristy. Protein Concentration Determination. http://www2.onu.edu/~k-
broekemeier/Chem314/ReviewProteinMethods.pdf (accessed September 12, 2019)

C. BONUS: What happened after the protagonist came back in his hometown in The Count of
Monte Cristo?