objectives

Protein assay methods

Protein assays are laboratory studies that are performed to measure the rate of enzyme activity for a biological process. Recall that enzymes are catalysts, so they are not consumed in a chemical reaction, but are regenerated, and a single enzyme molecule can perform catalysis for many substrate molecules. Although the concentration of the enzyme affects the rate of reaction, the concentration of the enzyme is generally not used to determine the rate of reaction, since they are not consumed and would show a consistent concentration throughout the reaction. Rather, the concentration of the substrate or the product(s) formed are generally studied to measure enzyme activity, as the substrate concentration decreases over time and the product concentration increases over time.

Common methods for monitoring rate of reaction include changes in the colour, conductivity or pH of the species involved in the reaction or measuring gas production. Since the concentrations involved are generally very small, the method used to monitor these small changes and equate them to concentration must be very precise.

Qualitative tests for proteins

Many biological molecules are coloured or can bind to coloured substances, so monitoring colour change is an easy method to determine the rate of reaction. If the reactant is coloured, then the decrease in the intensity of the colour can be examined over time. If the product is coloured, then the increase in the intensity of the colour can be examined over time. Qualitative colour changes can be used by comparing the sample to a reference, but often the changes are too small for the eye to distinguish, so a variety of instruments can be used to quantify the colour changes and more precisely equate them to a concentration. Additionally, some compounds absorb light in the ultraviolet range, which is not detected by the human eye, so instruments must be used to measure these colour changes.

A common method to test for the presence and quantity of proteins is with a Biuret test. A Biuret test involves adding a solution made of sodium hydroxide, copper (II) sulfate and potassium sodium tartrate, commonly known as Biuret reagent. This solution is blue in colour, due to the presence of Cu2+ ions, but turns violet in the presence of peptide bonds, indicating the presence of proteins. The colour change can be qualitatively or quantitatively assessed to determine both the presence and the concentration of protein present in a solution sample.

Quantitative tests for proteins

To quantitatively determine the concentration of proteins from a Biuret test, spectroscopy is generally used. Coloured compounds absorb light of a particular wavelength, so the specific wavelength that a substance will absorb must first be identified. Instruments, such as a colorimeter, are used to determine which wavelength is absorbed and how much light is absorbed, equating absorbance to concentration.

A colorimeter is an instrument that uses a filter applied to a source of white light. The filter causes only a single wavelength of light, referred to as monochromatic light, to pass through the sample and to a detector, which determines if any light was absorbed. The sample is contained in a specialised container, known as a cuvette, which can be made of plastic or glass that contains no blemishes and must be wiped clean of any dust or fingerprints to ensure that the material of the cuvette does not interfere with the light travelling through the sample. The width of the cuvette and sample can affect the absorbance, as more absorbance would occur if the light is passing through a greater quantity of sample. The path length refers to the width of the sample that the light must pass through in the cuvette. Often instruments use standard cuvettes of 1 cm.

Determining lambda max, λmax

Colorimeters must scan for each wavelength individually to see which wavelength has the greatest absorption, known as lambda max, λmax. Similar instruments can also scan for wavelengths in both the visible and ultraviolet ranges, known as UV-Vis Spectroscopy. Often, a reference sample is also scanned parallel to the sample. The reference, sometimes known as a 'blank', would contain the same solvent as the sample, but not the protein that is undergoing the test. This practice is done to ensure any light absorption from the cuvette or the solvent is accounted for and subtracted by the detector.

The data collected is then expressed as a graph of absorbance versus wavelength. Absorbance is a comparison of the intensity of the light before and after it passes through the sample. Absorbance has no units, but it is measured relative to the reference, which is designated an absorbance of 0.

Although there will be absorbance at a variety of wavelengths, there will generally only be one wavelength that shows the strongest absorbance. This wavelength of the highest absorbance is known as λmax.

Constructing a calibration curve

Once λmax is determined, the concentration of the protein in the sample can be measured. Absorbance and concentration of a substance have a directly proportional relationship, as determined by the Beer-Lambert Law. The Beer-Lambert law states that at low concentrations, there is a strong linear correlation between concentration and absorbance. The relationship loses its linear proportionality, however, at high concentrations, so this method of equating absorbance to concentration only works for low concentrations.

This strong linear relationship allows for the construction of a calibration curve to determine protein concentration. A series of reference standards at different, known concentrations can be made and tested for absorbance. The concentration and absorbance can be plotted on a graph and a line of best fit can be made. The sample of unknown concentration can then be measured for absorbance and the concentration determined from the graph.

The Beer-Lambert law is generally expressed mathematically as:

where ε is the molar extinction coefficient, a constant that is unique to each substance, and l is the path length, the thickness of the sample and cuvette. Since calibration curves are constructed with absorbance on the y-axis and concentration on the x-axis, A = εlc can be expressed as a linear equation in the form y = mx+b.

The line of best fit on a calibration curve will always pass through the origin (0,0), so there is no 'b' term and the path length will always be constant for the same instrument, generally 1 cm. With this equation, the slope of the line, m, will be equal to ε, the molar extinction coefficient.

For example, in Figure 6, we can calculate the slope using two points, the origin (0,0) and the last point (5.0, 0.40).

Therefore, the value of the molar extinction coefficient is 0.08. This value can sometimes be used to identify a substance, since all coloured substances have a unique molar extinction coefficient value.