Protein analysis: determination of total protein

• Crude protein analysis using the Kjeldahl method
• Improved Dumas method
• Methods using UV-visible spectroscopy
  • Direct measurement at 280nm
  • Biuret method
  • Lowry Method
  • Dye binding methods
  • Turbimetric method
  • Advantages & Disadvantages
• Other instrumental techniques
  • Measurement of bulk physical properties
  • Measurement of adsorption of radiation
  • Measurement of scattering of radiation
• Comparison of methods

Kjeldahl analysis

The crude protein analysis using the Kjeldahl method is explained in the section 'Proximate Analysis'.

Improved Dumas method

The Dumas method, which was abandoned long ago in favor of the Kjeldahl method, is making a comeback in a new and vastly improved form. This automated instrumental technique capable of rapidly measuring the protein concentration of food samples. It is based on a method first described by a scientist called Dumas over a century and a half ago. It is beginning to compete with the Kjeldahl method as the standard method of analysis for proteins for some foodstuffs due to its rapidness.

Principle

A sample of known mass is combusted in a high temperature (about 900 ^oC) chamber in the presence of oxygen. This leads to the release of CO₂, H₂O and N₂. The CO₂ and H₂O are removed by passing the gasses over special columns that absorb them. The nitrogen content is then measured by passing the remaining gasses through a column that has a thermal conductivity detector at the end. The column helps separate the nitrogen from any residual CO₂ and H₂O 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, such as EDTA (= 9.59%N). Thus 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 in a sample to the protein content, using suitable conversion factors which depend on the precise amino acid sequence of the protein.

Advantages: It is much faster than the Kjeldahl method (under 4 minutes per measurement, compared to 1-2 hours for Kjeldahl). It doesn't need toxic chemicals or catalysts. Many samples can be measured automatically. It is easy to use. The costs per analysis are lower than for Kjeldahl analysis.
Disadvantages: High initial cost. It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. Different proteins need different correction factors because they have different amino acid sequences. The small sample size makes it difficult to obtain a representative sample.

The combustion technique for crude protein has received AOAC approval for cereal grains and oilseeds, meat/meat products, and animal feed. It is currently being reviewed for dairy products.

Methods using UV-visible spectroscopy

A number of methods have been devised to measure protein concentration, which are based on UV-visible spectroscopy. These methods use either the natural ability of proteins to absorb (or scatter) light in the UV-visible region of the electromagnetic spectrum, or they chemically or physically modify proteins to make them absorb (or scatter) light in this region. The basic principle behind each of these tests is similar. First of all a calibration curve of absorbance (or turbidity) versus protein concentration is prepared using a series of protein solutions of known concentration. The absorbance (or turbidity) of the solution being analyzed is then measured at the same wavelength, and its protein concentration determined from the calibration curve. The main difference between the tests are the chemical groups which are responsible for the absorption or scattering of radiation, e.g., peptide bonds, aromatic side-groups, basic groups and aggregated proteins.

Direct measurement at 280nm

Tryptophan and tyrosine absorb ultraviolet light strongly at 280 nm. The tryptophan and tyrosine content of many proteins remains fairly constant, and so the absorbance of protein solutions at 280nm can be used to determine their concentration. The advantages of this method are that the procedure is simple to carry out, it is nondestructive, and no special reagents are required. The major disadvantage is that nucleic acids also absorb strongly at 280 nm and could therefore interfere with the measurement of the protein if they are present in sufficient concentrations. Even so, methods have been developed to overcome this problem, e.g., by measuring the absorbance at two different wavelengths.

Biuret Method

A violet-purplish color is produced when cupric ions (Cu²⁺) interact with peptide bonds under alkaline conditions. The biuret reagent, which contains all the chemicals required to carry out the analysis, can be purchased commercially. It is mixed with a protein solution and then allowed to stand for 15-30 minutes before the absorbance is read at 540 nm. The major advantage of this technique is that there is no interference from materials that adsorb at lower wavelengths, and the technique is less sensitive to protein type because it utilizes absorption involving peptide bonds that are common to all proteins, rather than specific side groups. However, it has a relatively low sensitivity compared to other UV-visible methods.

Lowry Method

The Lowry method combines the biuret reagent with another reagent (the Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins. This gives a bluish color which can be read somewhere between 500 - 750 nm depending on the sensitivity required. There is a small peak around 500 nm that can be used to determine high protein concentrations and a large peak around 750 nm that can be used to determine low protein concentrations. This method is more sensitive to low concentrations of proteins than the biuret method.

Dye binding methods

A known excess of a negatively charged (anionic) dye is added to a protein solution whose pH is adjusted so that the proteins are positively charged (i.e. < the isoelectric point). The proteins form an insoluble complex with the dye because of the electrostatic attraction between the molecules, but the unbound dye remains soluble. The anionic dye binds to cationic groups of the basic amino acid residues (histidine, arganine and lysine) and to free amino terminal groups. The amount of unbound dye remaining in solution after the insoluble protein-dye complex has been removed (e.g., by centrifugation) is determined by measuring its absorbance. The amount of protein present in the original solution is proportional to the amount of dye that bound to it: dye_bound = dye_initial - dye_free.

Turbimetric method

Protein molecules which are normally soluble in solution can be made to precipitate by the addition of certain chemicals, e.g., trichloroacetic acid. Protein precipitation causes the solution to become turbid. Thus the concentration of protein can be determined by measuring the degree of turbidity.

Advantages & Disadvantages

Advantages: UV-visible techniques are fairly rapid and simple to carry out, and are sensitive to low concentrations of proteins.

Disadvantages: For most UV-visible techniques it is necessary to use dilute and transparent solutions, which contain no contaminating substances which absorb or scatter light at the same wavelength as the protein being analyzed. The need for transparent solutions means that most foods must undergo significant amounts of sample preparation before they can be analyzed, e.g., homogenization, solvent extraction, centrifugation, filtration, which can be time consuming and laborious. In addition, it is sometimes difficult to quantitatively extract proteins from certain types of foods, especially after they have been processed so that the proteins become aggregated or covalently bound with other substances. In addition the absorbance depends on the type of protein analyzed (different proteins have different amino acid sequences).

Other instrumental techniques

There are a wide variety of different instrumental methods available for determining the total protein content of food materials. These can be divided into three different categories according to their physicochemical principles: (i) measurement of bulk physical properties, (ii) measurement of adsorption of radiation, and (iii) measurement of scattering of radiation. Each instrumental methods has its own advantages and disadvantages, and range of foods to which it can be applied.

Measurement of Bulk Physical Properties

• Density: The density of a protein is greater than that of most other food components, and so there is an increase in density of a food as its protein content increases. Thus the protein content of foods can be determined by measuring their density.
• Refractive index: The refractive index of an aqueous solution increases as the protein concentration increases and therefore RI measurements can be used to determine the protein content.

Measurement of Adsorption of Radiation

• UV-visible: The concentration of proteins can be determined by measuring the absorbance of ultraviolet-visible radiation (see above).
• Infrared: Infrared techniques can be used to determine the concentration of proteins in food samples. Proteins absorb IR naturally due to characteristic vibrations (stretching and bending) of certain chemical groups along the polypeptide backbone. Measurements of the absorbance of radiation at certain wavelengths can thus be used to quantify the concentration of protein in the sample. IR is particularly useful for rapid on-line analysis of protein content. It also requires little sample preparation and is nondestructive. Its major disadvantages are its high initial cost and the need for extensive calibration.
• Nuclear Magnetic Resonance: NMR spectroscopy can be used to determine the total protein concentration of foods. The protein content is determined by measuring the area under a peak in an NMR chemical shift spectra that corresponds to the protein fraction.

Measurement of Scattering of Radiation

• Light scattering: The concentration of protein aggregates in aqueous solution can be determined using light scattering techniques because the turbidity of a solution is directly proportional to the concentration of aggregates present.
• Ultrasonic scattering: The concentration of protein aggregates can also be determined using ultrasonic scattering techniques because the ultrasonic velocity and absorption of ultrasound are related to the concentration of protein aggregates present.

Advantages and Disadvantages

A number of these instrumental methods have major advantages over the other techniques mentioned above because they are nondestructive, require little or no sample preparation, and measurements are rapid and precise. A major disadvantage of the techniques which rely on measurements of the bulk physical properties of foods are that a calibration curve must be prepared between the physical property of interest and the total protein content, and this may depend on the type of protein present and the food matrix it is contained within. In addition, the techniques based on measurements of bulk physicochemical properties can only be used to analyze foods with relatively simple compositions. In a food that contains many different components whose concentration may vary, it is difficult to disentangle the contribution that the protein makes to the overall measurement from that of the other components.

Comparison of methods

As food scientists we may often be in a position where we have to choose a particular technique for measuring the protein concentration of a food. How do we decide which technique is the most appropriate for our particular application ? The first thing to determine is what is the information going to be used for. If the analysis is to be carried out for official purposes, e.g., legal or labeling requirements, then it is important to use an officially recognized method. The Kjeldahl method, and increasingly the Dumas method, have been officially approved for a wide range of food applications. In contrast, only a small number of applications of UV-visible spectroscopy have been officially recognized.

For quality control purposes, it is often more useful to have rapid and simple measurements of protein content and therefore IR techniques are most suitable. For fundamental studies in the laboratory, where pure proteins are often analyzed, UV-visible spectroscopic techniques are often preferred because they give rapid and reliable measurements, and are sensitive to low concentrations of protein.

Other factors which may have to be considered are the amount of sample preparation required, their sensitivity and their speed. The Kjeldahl, Dumas and IR methods require very little sample preparation. After a representative sample of the food has been selected it can usually be tested directly. On the other hand, the various UV-visible methods require extensive sample preparation prior to analysis. The protein must be extracted from the food into a dilute transparent solution, which usually involves time consuming homogenization, solvent extraction, filtration and centrifugation procedures. In addition, it may be difficult to completely isolate some proteins from foods because they are strongly bound to other components. The various techniques also have different sensitivities, i.e., the lowest concentration of protein which they can detect. The UV-visible methods are the most sensitive, being able to detect protein concentrations as low as 0.001 wt%. The sensitivity of the Dumas, Kjeldahl and IR methods is somewhere around 0.1 wt%. The time required per analysis, and the number of samples which can be run simultaneously, are also important factors to consider when deciding which analytical technique to use. IR techniques are capable of rapid analysis (< 1 minute) of protein concentration once they have been calibrated. The modern instrumental Dumas method is fully automated and can measure the protein concentration of a sample in less than 5 minutes, compared to the Kjeldahl method which takes between 30 minutes and 2 hours to carry out. The various UV-visible methods range between a couple of minutes to an hour (depending on the type of dye that is used and how long it takes to react), although it does have the advantage that many samples can be run simultaneously. Nevertheless, it is usually necessary to carry out extensive sample preparation prior to analysis in order to get a transparent solution. Other factors which may be important when selecting an appropriate technique are: the equipment available, ease of operation, the desired accuracy, and whether or not the technique is nondestructive.