1. Assessment of protein preparations
Don’t waste clean thinking on dirty enzymes
Today’s class goal
Determination of protein content
Assessment of protein activity

2. Characterization of purified proteins
After a purification procedure proteins can be obtained either in a form of pure solid protein or in a form of a solution of a given concentration.
In medicine, proteins are frequently administered in a form of solutions. So, it is convenient to produce proteins as a “ready-to-use” formulations

3. Quality control for protein preparations implies (1) the determination of target protein content, (2) assessment of a required activity of protein, (3) determination of impurities
(1) Quantitation is aimed at the demonstration of the proximity of protein content to 100 %
(2) For most medical, biotechnological, etc applications, a protein must be in its native, active form. The assessment of protein activity is the simplest way to estimate the content of native protein.
(3) Some impurities can compromise the quality of a protein formulations significantly. Therefore those key impurities must be revealed and quantified
Chemical analysis does not deliver information concerning the content of native protein. It is only quantify the percentage of a polypeptide corresponding to a given protein without disclosing its spacious structure. One hundred percent of right amino acids in a right sequence do not guarantee that the sample will exhibit 100 % activity, since a part of the sample can be in the denaturated state.

4. Quantitation of total protein
Protein content can be determined by using certain specific chemical or physical properties unique to proteins
Chemical methods
2. Physico-chemical methods
Determination
of total nitrogen
Spectrophotometry
Spectrofluorimetry

5. Determination of total nitrogen
There are 3 main methods to measure total nitrogen: Kjeldahl, Dumas, and combustion methods. The most popular is the method of Kjeldahl.
Kjeldahl method
In general, it is assumed that a pure protein will contain 16% nitrogen. Thus, the protein content of a sample is equal to the nitrogen content multiplied by a nitrogen-to-protein conversion factor, 6.25 (i.e., 100/16)
For the determination of proteins in food, specific conversion factor should be used that take into account nonprotein content of samples

6. Scheme of the measurement of nitrogen in the Kjeldahl method
The sample is digested in sulfuric acid, using CuSO,/TiO, as catalysts, converting N to NH3,

7. Scheme of the measurement of nitrogen in the Kjeldahl method
NH3 is distilled and titrated

8. Automatic digester for the Kjeldal method

9. Spectrophotometric assays
The method of spectrophotometry is based on the formation of a colored compound, whose concentration is determined by comparing the absorbance of a sample solution with the absorbance of a standard solution.
Selection of a standard protein is a sophisticated issue.
Options:
A highly purified predominate protein (Best choice. not always possible)
Protein that will produce a similar color response curve with the selected protein assay method (frequent choices are BSA or BGG)

10. Spectrophotometric assays
Bradford method
Biuret method
Lowry method
Bicinchoninic acid (BCA) method

11. 1. Bradford method
(c) Handbook of Food Analytical Chemistry. Wiley, 2005, Section B

12. 2. Biuret method Biuret reaction
The assay is based on the formation of colored chelate complex between amino acid residues and cupric ions in alkaline environment
Biuret reaction
lmax = 750 nm
(c) Handbook of Food Analytical Chemistry. Wiley, 2005, Section B

13. 3. Lowry method
A modified version of the biuret test.
BLUE
lmax = 750 nm
Chelate complexation of cupric ions with the nitrogen atoms of amino groups.
Oxidation of aromatic residues with Folin reagent
(c) Handbook of Food Analytical Chemistry. Wiley, 2005, Section B

14. 4. Bicinchoninic acid method
Reduction of Cu2+ by protein in an alkaline medium followed by the complexation of the cuprous cation (Cu+) by bicinchoninic acid
lmax = 562 nm
(c) Handbook of Food Analytical Chemistry. Wiley, 2005, Section B

15. Fluorimetric assays
Spectrofluorimetry is a spectrometric method, in which a sample is irradiated at one wavelength and emits light at another wavelength. Intensity of the emitting light can be measured. It is proportional to the concentration of an analyte
Fluorescent probes: NanoOrangeTM and Quant-iTTM
Fluorescent probes are bound to a proteins forming a fluorescent complex.

16. Interfering influence
Selection of assay method
Assay
Range
Interfering influence
Reducing compounds
Detergents
Bradford
1 – 50 mg +
Biuret
5,000 – 160,000 mg
Lowry
5 – 100 mg
BCA
0.2 – 50 mg ―
Fluorescent probes mg

17. Measurement of enzyme activity
Enzymes are catalysts. Their function in a cell is to make reactions faster.
Process of the transformation of a substrate S into a product P is intensified if an intermediate active complex Substrate – Enzyme (ES) is formed.
k+1, k-1, k+2 – rate constants

18. Example: Activity of fatty acid hydroperoxide lyase (HPO lyase) in the breakdown of fatty acid hydroperoxides
(c) M.P. Santiago-Gomez et al. / Enzyme and Microbial Technology 41 (2007) 13–18