Analysis+of+Proteins



Analysis of Proteins - Summer Wiki By: Paloma Santa Cruz, Fernando Dasso and Camila Medina

**__6.1 - Introduction: __**

 * As seen on previous sections, proteins are polymers of amino acids.
 * Up to 20 different types of amino acids occur naturally in proteins.
 * Proteins can be differentiated from each other by analysing the type and the number and sequence of amino acids that make the polypeptide backbone.
 * Hence, proteins have diverse molecular strutters, nutritional attributes and physiochemical properties.
 * Proteins are an important content of food and also used in food as ingredients.
 * Used as gelling agents, emulsifiers, foaming agents and thickeners
 * Many of these food proteins are enzymes capable of enhancing the rate of certain biochemical reactions, and these reactions can have either favourable or detrimental effect of the overall properties of food.
 * Food analysis focus in knowing the total concentration, type, molecular structure and functional properties of proteins in foods.

__**6.2 - Determination of Overall Protein Concentration **__
//6.2.1 - Kjeldahl method//
 * Developed in 1883 by a brewer called Johann Kjeldahl.
 * A food is digested with a strong acid -> it releases nitrogen which can be determined by a suitable titration technique.
 * The amount of protein present is calculated from the nitrogen concentration of the food.
 * This approach is used up until today but many arrangements have been made.
 * Considered to be the standard method of determining protein concentration.
 * The Kjeldahl method does not measure the protein content directly so a conversion factor is needed to convert the amount of nitrogen concentration to a protein concentration.
 * The conversion factor of 6.25 (equivalent to 0.16g nitrogen per gram of protein) is used, however,this is an average value and each protein has its own conversion factor.
 * This method can be divided into three steps: digestion, neutralisation and titration.

//6.2.1.1 - Principles//

N(food) ® (NH4)2SO4 (1) (NH4)2SO4 + 2 NaOH ® 2NH3 + 2H2O + Na2SO4 (2)
 * Digestion
 * The food sample is weighed into a digestion flask and then digested by heating it in the presence of sulfuric acid, anhydrous sodium sulcate and a catalyst such as copper, titanium or mercury.
 * The digestion converts any nitrogen in the food into ammonia and other organic matter to carbon dioxide and water.
 * Ammonia gas is not liberated in an acid solution because the ammonia is in the form of the ammonium ion which binds to the sulfate ion and thus remains in solution:
 * Neutralization
 * After the digestion has been completed, the digestion flask is connected to a receiving flask by a tube.
 * The solution in the digestion flask is made alkaline by the addition of sodium hydroxide, which converts the ammonium sulfate into ammonia gas:
 * The ammonia gas formed is left out from the solution and moves out of the digestion flask and into the receiving flask.
 * The low pH of the solution in the receiving flask converts ammonia gas to ammonium ion and the boric acid to borate ion.

NH3 + H3BO3 (boric acid) ® NH4+ + H2BO3- (borate ion) (3)
 * Titration

H2BO3- + H+ ® H3BO3 (4)
 * The nitrogen content is then estimated by titration of the ammonium borate formed above, using an indicator to determine the end-point of the reaction
 * The concentration of hydrogen ions (in moles) required to reach the end-point is equivalent to the concentration of nitrogen that was in the original food (equation 3)
 * The following equation can be used to find the nitrogen concentration of a sample that weighs m grams using a xM HCl acid solution for the titration:
 * Vs and Vb are the titration volumes of the sample and blank, and 14g is the molecular weight of nitrogen (N).
 * A blank sample is normally ran at the same time as the material being analysed to consider any residual nitrogen which may be in the reagents used to carry out the experiment.
 * Once the nitrogen content has been found it is converted to a protein content using the conversion factor.

//6.2.1.4 - Advantages and Disadvantages//
 * __Advantages:__
 * The method is widely used internationally and remains to be the standard method.
 * It is universal, precise and reproducible.
 * __Disadvantages:__
 * Does not give a measure of the true protein, since all nitrogen in food is not in the form of protein.
 * Different proteins need different conversion factors.
 * The use of concentrated sulfuric acid at high temperatures is not safe.
 * Technique is very time-consuming.

//6.2.2 - Enhanced Dumas Method//
 * Not long ago, an automated instrumental technique has been developed which is able to measure the protein concentration of food samples in reduced amounts of time.
 * This technique is based on a method first explained by a scientist (Dumas) over a century and a half ago.
 * It is starting to compete with the Kjeldahl method as the standard method of analysis for proteins for some foodstuffs due to its rapidness.

//6.2.2.1 - General Principles//
 * A sample of known mass is combusted in a high temperature (about 900 degrees C) chamber in the presence of oxygen.
 * This causes the release of carbon dioxide, water and N 2.
 * The carbon dioxide and water are removed by passing the gasses over special columns that absorb them.
 * The nitrogen content is measured by passing the remaining gasses through a column that has thermal conductivity detectors at the end.
 * The column helps separate the nitrogen from any residual that may have remained in the gas stream.
 * The instrument is calibrated by analyzing a material that is pure and has a known nitrogen concentration.
 * The signal from the thermal conductivity detector can be converted into a nitrogen content.
 * As with the Kjeldahl method it is necessary to convert the concentration of nitrogen to a protein content using the appropriate conversion factors.

//6.2.2.2 - Advantages and Disadvantages//


 * __Advantages:__
 * The "Enhanced Dumas Method" is much faster than the "Kjeldahl Method"
 * The time it takes to make measurements in this method is of approximately 4 minutes versus the 1 to 2 hours required in the Kjeldahl Method
 * No toxic chemicals or catalysts are required therefore, the method is safer
 * The measurements of many sample can be done automatically
 * The method is far simpler and thus, easier to use


 * __Disadvantages:__
 * The initial cost to complete this method is high
 * Because all the nitrogen found is foods is not in the form of a protein, using this method does not give a measure of the true protein
 * Since proteins have different sequences of amino acids, correction factors required for this method change based on the type of protein used
 * Due to the fact that the size of the sample taken is small, it is difficult to obtain a representative sample

//6.2.3 - Methods Using UV-Visible Spectroscopy//


 * **UV-visible spectroscopy** is used as the base of different methods that measure protein concentration
 * These methods either rely on the ability of proteins to absorb light in the UV-visible region of the electromagnetic spectrum, or they modify proteins (chemically or physically) to make them absorb light in this region
 * The process of all methods is similar and as follows:
 * __First:__ Protein solutions of known concentrations are used to create a **calibration curve of absorbance** (also called turbidity) **versus a protein concentration**
 * __Second:__Using the calibration curve the protein concentration of the solution under analysis is determined, and the absorbance of this same solution is measured at the same wavelength
 * It is important to know that the difference between the methods are the chemicals groups of the solutions being analyzed, which are responsible for the absorption of radiation
 * eg: peptide bonds, aromatic side-groups, basic groups, and aggregated proteins

//6.2.3.1 - Principles//
 * Direct Measurement at 280nm
 * At 280nm both //tryptophan and tyrosine// absorb UV light
 * Because the content of both elements mentioned above remains fairly constant in many proteins, the absorbance of protein solutions at 280nm can be used to determine their concentration
 * Advantages of this method:
 * Procedures is simple
 * Method is nondestructive
 * No special reagents are required
 * Disadvantages of this method:
 * At 280nm nucleic acids also absorb UV light and can thus interfere with the measurement of the protein if in the process they are present at sufficient concentrations
 * But there is a way to solve this problem which is by measuring the absorbance of the proteins at two different wavelengths


 * Biuret Method
 * When cupric ions (Cu 2+ )  interact with //peptide bonds// under alkaline solutions, a violet-purplish color is produced
 * The biuret reagent containing the chemicals required to carry out the analysis, is then mixed with the protein solution and left between 15-30 minutes after which the absorbance is taken at 540nm
 * Advantages of this method:
 * No interference with the measurements because of the materials that absorb at lower wavelengths
 * Method is less sensitive to protein type because instead of specific side groups, it uses absorption involving peptide bonds that are universal to all proteins
 * Disadvantage of this method:
 * When compared to other UV-methods, this one has a relatively low sensitivity

dye bound = dye initial - dye free.
 * Lowry Method
 * Combines the biuret reagent with another reagent (called the **Folin-Ciocalteau Phenol Reagent)** to react with the //tyrosine// and the //trytophan// residues found in the proteins producing a bluish color that is between a 500-750nm frequency reading depending on the sensitivity required
 * The high peak at 500nm can be used to determine high protein concentrations
 * The low peak at 750nm can be used to determine low protein concentrations
 * Major Disadvantage:
 * Greater sensitivity to low concentrations of proteins than the "Biuret Method"
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 13px; line-height: 1.5;">Dye Binding Methods
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 13px; line-height: 1.5;">Negatively charged dye (called **anionic****)** is added to a protein solution whose pH is adjusted so that the proteins become positively charged (**cationic**)
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 13px; line-height: 1.5;">The proteins form an **insoluble** **complex** with the dye because of the attraction between the molecules
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 13px; line-height: 1.5;">The unbound dye remains **soluble**
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 13px; line-height: 1.5;">The anionic dye binds to the cationic groups of amino acid residues and free amino terminal groups
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 13px; line-height: 1.5;">After the //**insoluble protein-dye complex**// is removed the residue of unbound dye in the solution is determined by measuring the absorbance
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 13px; line-height: 1.5;">The amount of protein in the original solution is proportional to the amount of dye bound to it:


 * Turbimetric Method
 * Protein molecules are made into precipitate using certain chemicals like //trichloroacetic acid//
 * This causes the solution to become turbid because proteins in a solution are usually soluble
 * Therefore, the concentration of the protein can be determined by measuring the degree of turbidity in the solution

//6.2.3.2 - Advantages and Disadvantages//


 * __Advantages:__
 * The UV-Visible Method has techniques that are fast and simple to complete
 * These techniques are also sensitive to low concentrations of proteins


 * __Disadvantages:__
 * Most UV techniques require dilute and transparent solutions that contain no contaminating substances which wither absorb or scatter light at the same wavelength as the protein being analyzed
 * Because transparent solutions are used this suggests that foods must go through many amounts of sample preparations before they can be analyzed, taking more time and work:
 * They can undergo:
 * homogenization
 * solvent extraction
 * filtration
 * centrifugation
 * In this method it is also hard to extract proteins quantitatively from certain types of foods, especially after these foods have been processed causing proteins to covalently bond with other substances
 * Absorbance depends on the protein used for analysis because each protein contains a different sequence of amino acids

//6.2.4 - Other Instrumental Techniques//


 * For calculating the total protein content of foods, different instrumental methods can be used
 * These are divided into three categories according to //**psychochemical principles:**//
 * 1). Measurement of Bulk Physical Properties
 * 2). Measurement of Absorption of Radiation
 * 3). Measurement of Scattering of Radiation
 * Each method has its advantages and disadvantages, as well as a range of foods to which it can be applied

//6.2.4.1 - Principles//


 * Measurement of Bulk Physical Properties
 * **Density:**
 * The density of a protein is greater than that of most of the other food components
 * So if the protein content increases so does the density of the food
 * Therefore, protein content of foods can be calculated by measuring the density of the food
 * **Refractive Index (RI):**
 * The refractive index (RI) of an //aqueous solution// increases as the protein concentration rises
 * So, the RI can be used to determine the protein content


 * Measurement of Absorption of Radiation
 * **UV-Visible:**
 * The concentration of proteins can be determined by measuring the absorbance of UV-visible radiation
 * **Infrared Techniques (IR):**
 * These techniques are used to determine the protein concentration within food samples
 * IR is absorbed naturally by proteins via characteristic vibrations (stretching and bending) of certain chemical groups along the polypeptide backbone
 * Therefore, the measurements taken of the absorbance of radiation at different wavelengths can be used to determine the concentration of protein in a sample
 * ADVANTAGES:
 * Useful in rapid on-line analysis of protein content
 * Requires little preparation
 * Process and method is nondestructive
 * DISADVANTAGES:
 * High initial cost
 * Need of extensive calibration
 * **Nuclear Magnetic Resonance (NMR):**
 * This technique can be used to determine the total concentration of proteins in foods
 * This is done by measuring the area under the peak in a NMR chemical shift spectra that corresponds to the protein under analysis


 * Measurement of Scattering of Radiation:
 * ** Light Scattering: **
 * The concentration of protein aggregates in an //aqueous solution// can be determined using light scattering techniques because the turbidity of a solution is directly proportional to the concentration of protein aggregates present
 * **Ultrasonic Scattering:**
 * The concentration of protein aggregates can be determined also by using ultrasonic scattering techniques because the velocity and absorption of the ultrasound are related to the concentration of protein aggregates present

//6.2.4.2 - Advantages and Disadvantages//


 * __ Advantages: __
 * These instrumental methods have more advantages when compared to the other methods because they are:
 * Nondestructive
 * Require little or no sample preparation
 * Measurements are fast and precise


 * __Disadvantages:__
 * Major disadvantage of the techniques that rely on the measurements of the bulk physical properties of foods are:
 * A calibration curve must be prepared between the physical property of interest and the total protein content
 * Therefore, this may rely on the type of protein present and the food matrix that is contained within
 * Techniques that rely on these measurements can only be used to analyze foods that have very simple compositions because when a food has different components whose concentrations may vary, it is difficult to determine the contribution that one protein makes to the overall measurement from that of the other components found within the food

<span style="font-family: 'Comic Sans MS',cursive;">Sources: http://kidshealth.org/kid/stay_healthy/body/headers_86064/K_proteins.gif http://people.umass.edu/~mcclemen/581Proteins.html