Prepared by
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111

Table of Contents


To analyze amino acid concentrations by the ninhydrin colorimetric method during the enzymatic hydrolysis of a protein.


    H H O H H O H H O H H O                H H O H H O          O H H O
    | | = | | = | | = | | =      H2O       | | = | | =          = | | =
...-N-C-C-N-C-C-N-C-C-N-C-C-... -----> ...-N-C-C-N-C-C-OH + NH2-C-C-N-C-C-...
      |     |     |     |                    |     |            |     |
      R1    R2    R3    R4                   R1    R2           R3    R4

Figure 1. The breakdown of protein through the hydrolysis of peptide bonds
Various species of organisms cannot synthesize or are not efficient in generating all of the twenty amino acids needed to construct the proteins and enzymes essential for their survival. To sustain growth and to maintain metabolic functions, these amino acids must be provided from outside sources. This can be accomplished by the intake of proteins. Humans are a good example of living organisms that ingest proteins as part of their nutritional requirements. However, protein molecules are generally quite large, and these large molecules cannot be transported across cell membranes for the same reason that you cannot bring an elephant through the door into your dorm room. Some organisms secrete proteolytic enzymes extracellular to break down the protein to its component monomeric amino acid units by hydrolyzing peptide bonds at the end of the polymer chain. A series of shorter polypeptides of different lengths are also formed if the broken peptide bonds are not at the end of the polymer chain. Thus, depending on the location of the attack, proteases can be further classified into exopeptidases (attack on the terminal group) and endopeptidases (attack on internal linkages).

In the previous experiment, the hydrolysis of a protein was monitored with the release of a dye that was bound to the protein. In this experiment, another more accurate and generally accepted color method is introduced. In this method, an organic compound called ninhydrin is reacted with the amino acids released during the hydrolysis of the protein. The original unreacted ninhydrin is yellowish in color, but the reacted product of ninhydrin has a deep purple-blue color. For example, the procedure given at the end of this section yields an absorbance of 0.27 for 1.X10^-4M of glutamic acid. Since ninhydrin does not react with the undegraded protein, one can measure the amino acid concentration by following the development of the purple color by measuring the absorbance of the solution with a spectrophotometer. Because the color intensity is a measure of the amino acid present, the color should intensify as more protein is degraded to amino acid over time. The upper limit in color intensity is reached when all the ninhydrin originally present in the solution has been consumed. Thus, the amount of ninhydrin originally present in the reaction mixture determines the maximum amino acid concentration that can be detected.

List of Reagents and Instruments

A. Equipment

B. Reagents


  1. Make a 10g/liter protein mixture by dissolving casein (Sigma) in water. (See Notes 1. and 3.)
  2. Make a saturated protease solution by adding 0.5 g of powder protease to 1 liter of water. Spin the solution in a centrifuge to separate the undissolved powder. Keep the supernatant. (See Note 2.)
  3. Mix equal volumes of protein solution and protease solution obtained in Step 1 and Step 2. Note the time at the start of the hydrolysis reaction.
  4. Withdraw 5 ml of the solution and measure the amino acid concentration of the solution as a function of time by using the ninhydrin colorimetric method. Suggested sampling time interval: 10 minutes for at least one hour.
  5. If time permits, repeat the same procedures for hair.


  1. Gelatin and albumin may be used in lieu of casein. The hydrolysis rate for different substrates may be studied.
  2. Alternatively, add 0.05 g protease directly to 50 ml of the protein mixture. However, one must perform the ninhydrin test on the supernatant obtained either through centrifugation or filtration for each sample.
  3. If reagent grade casein is not available, a 10g/l mixture can be made by the following steps:
    • Add 1 g of protein (cheese from the previous experiment or from a to 70 ml of water; using a blender, "liquefy" the mixture; pour into a 100 ml graduated cylinder or a 100 ml volumetric flask.
    • Rinse the blender with about 20 ml of water from a squeezable plastic bottle; pour the rinse into the measurement device in Step 3a; add water to 100 ml.


This experiment will also introduce students to the fundamentals of quantitative assays. First, when a spectrophotometer is used to quantitatively measure the absorbance of a colored solution, an absorbance spectrum should be obtained to determine the best wavelength to use. Thereafter, the same wavelength is used for all the subsequent determinations. Secondly, when there is a gradual development of color due to the limited reaction rate of the color reaction, the time needed for the completion of the reaction should be determined from the plot of absorbance versus time. Obviously, this time is when the change in the absorbance as a function of time is no longer significant. Thirdly, a calibration curve must be obtained by subjecting standard solutions of known concentrations to the same procedure so that the absorbance measurement can be correlated to actual physical units, in this case, the amino acid concentration. Finally, one should always remember to compare his sample to some reference, in this case, the reference being the protein mixture with no protease added. To obtain a blank reading, add ninhydrin reagent to the protein solution in the absence of protease and find the absorbance from the ninhydrin reaction. Protein solution in the absence of protease may not always give absolutely negative results due to other contaminants present in the solution, or the protein itself may contribute to the development of purple color to a certain degree.


  1. Report the absorption spectrum of the ninhydrin -- amino acid mixture. Which wavelength did you choose to measure the amino acid concentration? Why?
  2. How much time was needed to develop the color fully? Justify your answer with experimental data. (It should be 4-7 minutes.)
  3. Report your findings on protein digestion as a function of time. Explain what you have observed. Also calculate the enzyme activity and the rate of hydrolysis.
  4. What amino acids can human cells manufacture? What amino acids must be provided from outside sources? Can a well balanced amino acid diet replace the human intake of all the proteins?
  5. What are the amino acid contents of a typical protein? Express your answer in weight percent and in relative number of occurrences.
  6. Assuming that all the peptide bonds are equally susceptible to protease attacks and given that the starting protein has a well defined number of amino acid sub-units, say 1000, find the distribution of chain length as a function of reaction time. What is the free amino acid concentration as a function of time. Assume a 0th order reaction mechanism, say 100 bonds per minute per unit of enzyme. (Consult references on probability theories if you have difficulties in answering this question.)
  7. Repeat the above calculation for a system in which the starting protein has a given size (chain length) distribution, say a normal (Gaussian) distribution with an average of 1000 and a standard deviation of 100. Quantitatively how would your answer be affected if the bonds near the end of the chain were more susceptible to enzyme attacks than those away from the end? For this last part, assume an enzyme activity of 100 bonds per minute per unit of enzyme for the last bond, exponentially decreasing to half of that original value at 30th bonds away from the end. In the following figure, each AA represents an amino acid monomer unit.
                 1   2   3   4    26  27  28  29  30
    activity    100               50
  8. Do proteolytic enzymes attack themselves, given that the enzyme themselves are protein? If no, what make them special? If yes, what can be done to prolong their activities?
  9. Comment on ways to improve the experiment.


  1. James E. Bailey and David F. Ollis, Biochemical Engineering Fundamentals, 2nd Ed., p172-174, McGraw-Hill, 1986.
  2. Albert L. Lehninger, Biochemistry, p99, Worth Pub., 1975.

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Digestion of Protein into Amino Acid
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Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
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