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

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

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.

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

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

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,

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

Automatic digester for the Kjeldal method

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)

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

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

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

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

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

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.

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 0.02-2 mg

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

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