Biochemistry and Human Nutrition

Module 1. Bio-molecules

Lesson 6
SPECTROPHOTOMETRIC ASSAYS OF BIO-MOLECULES

6.1 Introduction

• Spectroscopy is a technique that measures the interaction of molecules with electromagnetic radiation. Light in the near-ultraviolet (UV) and visible (vis) range of the electromagnetic spectrum has an energy of about 150– 400 kJ /mol.
• The energy of the light is used to promote electrons from the ground state to an excited state. A spectrum is obtained when the absorption of light is measured as a function of its frequency or wavelength.
• Molecules with electrons in delocalized aromatic systems often absorb light in the near-UV (150–400 nm) or the visible (400–800 nm) region.
• Absorption spectroscopy is usually performed with molecules dissolved in a transparent solvent, such as in aqueous buffers.
• The absorbance of a solute depends linearly on its concentration and therefore absorption spectroscopy is ideally suited for quantitative measurements.
• The wavelength of absorption and the strength of absorbance of a molecule depend not only on the chemical nature but also on the molecular environment of its chromophores.
• Absorption spectroscopy is therefore an excellent technique for following ligand-binding reactions, enzyme catalysis and conformational transitions in proteins and nucleic acids. Spectroscopic measurements are very sensitive and nondestructive, and require only small amounts of material for analysis.

6.2 Lambert–Beer law

• In this technique, the amount of light that a sample absorbs at a particular wave length is measured and used to determine the concentration of the sample by comparison with appropriate standards or reference data.
• The most useful measure of light absorption is the absorbance (A), also commonly called the optical density (OD). The absorbance is defined as A = log I0 / I where I0 is the intensity of light that is incident on the sample and I is the intensity of light that is transmitted by the sample.
• The absorbance of a sample can be related to the concentration of the absorbing species through Beer's law:

A = ε cl

Where c is concentration, usually measured in moles per liter;
l is the length of the light path, usually 1 cm;
ε is a proportionality constant known as the molar extinction coefficient, with the units of liters per mole per centimeter.
The value of ε is a function of both the particular compound being measured and the wavelength.

Fig. 6.1 Beer's law for optical density

• Chlorophylls typically have an ε value of about 100,000 L mol^–1 cm^–1. When more than one component of a complex mixture absorbs at a given wavelength, the absorbances due to the individual components are generally additive.

6.3 The Spectrophotometer

The absorbance is measured by an instrument called a spectrophotometer. The essential parts of a spectrophotometer include a light source, a wavelength selection device such as a monochromator that contains a wavelength selection device such as a prism or filter, a sample chamber, a light detector such as a photomultiplier tube or silicon diode, and a readout device, usually also include a computer, which is used for storage and analysis of the spectra. The most useful machines scan the wavelength of the light that is incident on the sample and produce, as output, spectra of absorbance versus wavelength.

Fig. 6.2 Components of spectrophotometer

6.4 Estimation of DNA

DNA (Deoxyribonucleic Acid) is present in the nucleus of all cells. It is a double stranded molecule, made up of a chain of units called nucleotides and is a repository of genetic information.

6.4.1 Principle

Based on the reaction of deoxyribose sugar with diphenylamine reagent. Under extreme acidic conditions, the deoxyribose moiety of DNA is dehydrated and forms an aldehyde product ω-hydroxylevulinic acid, which condenses in acidic medium with diphenykamine to produce deep-blue coloured products, having absorption maxima at 595 nm. The colour produced is stable for several hours.

6.4.2 Notes

• This method is commonly employed for samples of 50-500 μg DNA.
• In the original method by Dische, acetaldehyde is not added. In Burton’s modified method, acetaldehyde is added, as it potentiates colour development and makes the method 3.5 times more sensitive than the original method.
• In DNA, only the deoxyribose of the purine nucleotides reacts, so that the value obtained represents half of the total deoxyribose present.
• Standard DNA solution is prepared in 0.1N NaOH, because this helps in dissolving the DNA, resulting in a clear solution.
• Glacial acetic acid is added to increase the rate of colour development.

6.5 Estimation of RNA

RNA (ribonucleic acid) is found in the cytoplasm of cells. There are three major classes of RNA (messenger RNA), tRNA (transfer RNA) and rRNA (ribosomal RNA). Ribosomal RNA constitutes the larger percentage of total RNA.

6.5.1 Principle

Estimation of RNA is carried out by the reaction of ribose in RNA with orcinol (Bial’s test). The method depends upon the conversion of the pentose sugar, Ribose to furfural, in the presence of hot acid. Furfural then reacts with orcinol, in the presence of ferric ions (Fe⁺⁺⁺⁾ to yield a green colour. The colour formed depends largely upon the concentration of hydrochloric acid, ferric chloride, orcinol and the time for which the solution is heated to 1000C.

6.5.2 Notes

1. Apart from orcinol, other reagents e.g. phloroglucinol, aniline etc. are also used for RNA estimation. However orcinol method (Bial’s test) is widely used, because in this method interference by DNA is only 0.85 % compared to 12% by other methods as given in literature.
2. Evaporation may be minimized by employing glass-stoppered tubes or by covering mouths of tubes with carefully cleaned marbles.
3. In the determination of RNA by Bial reaction, only the purine bound sugars react significantly.
4. The green color developed, if clear, is read at 660 nm, against blank. If turbid, extract with 5 ml of isoamyl alcohol and then read.
5. Xylose or adenylic acid can also be used as standard.
6. Dilute using n-butanol if the concentration of sample is high.
7. The yield and purity of RNA preparation can be assessed by measuring the absorbance of UV light by a solution of RNA. A pure solution should give a 260 nm: 280 nm of 2; one unit of A260 measured in 1 cm path length is equivalent to 40 μg/ml.

6.6 Estimation of Proteins

Proteins can be estimated by a number of methods e.g. Kjeldahl method, Nessler reaction, Biuret method, Ninhydrin reaction, Lowry method, UV absorbtion etc. Each method has its advantages and disadvantages. The choice of method eventually depends upon nature of sample, number of samples and ease of performance of assay. The Lowry’s method is the most commonly used method of protein estimation. It is easy to perform and is very sensitive (useful range is 0.005-0.02 mg protein). However it has got two major disadvantages- the intensity of colour varies with different proteins and the colour developed is not always proportional to concentration at higher values. Estimation of protein content of enzyme extracts is usually does by this method.

6.6.1 Estimation – Lowry (Folin-Ciocalteau) method

• The first step in the reaction involves the formation of a copper-protein complex in alkaline solution. This complex then reduces a phosphomolybdic-phosphotungstate reagent (Folin’s reagent) to yield an intense blue colour.
• Phenols are capable of reducing molybdenum in a complex of phosphomolybdotungstic acid. The tyrosine and tryptophan residues of proteins provide phenolic groups and cupric ions enhance the sensitivity. Thus, when treated with Folin-Ciocalteau’s reagent, proteins produce blue colour in varying degrees depending upon their tyrosine content. Hence, different proteins give different colour values.
• The blue colour produced by reduction of phosphomolybdotungstic acid by phenolic groups of the amino acids, tryptophan present in proteins plus the colour developed by the biuret reaction of the proteins with alkaline cupric tartarate are measured in Lowry’s method.
• The precaution to be observed when performing the assay concerns the addition of Folin’s reagent. This reagent is stable only at acidic pH; however the reduction reaction mentioned above occurs only at pH 10. Therefore, when Folin’s reagent is added to the alkaline copper-protein solution, mixing must occur immediately so that the reduction can proceed before the phosphomolybdic-phosphotungstate reagent breaks down.

6.6.2 Biuret method

Compounds with two or more peptide bonds give a violet colour with alkaline copper sulphate solution with absorbtion maxima at 540-560 nm. There is no interference from free amino acids and there is little dependence on amino acid composition as the copper reagent reacts with peptide chain itself rather than with side groups. The main disadvantage of this method is its low sensitivity – 1-6mg protein/ml, which severely limits its applicability.

6.6.3 Note

1. A linear relationship between amount of protein and colour intensity following treatment with the Folin-Ciocalteau reagent is observed only over a relatively limited range of protein concentration. A sample of unknown protein concentration should therefore be diluted above the upper limit of calibration curve.
2. A suggested range for establishing the curve is 0.01-0.20 mg (10-200 µg) of protein/tube.
3. For complete enzyme extraction, sometimes chemicals like EDTA, magnesium salts and mercaptoethanol are included, which interfere with the estimation. Hence, when these are present, the proteins should be precipitated by adding 10% TCA (trichloroacetic acid), centrifuged and the precipitate dissolved in 1N NaOH, before proceeding for protein estimation.
4. Rapid mixing as the Folin-Ciocalteau reagent is added is important for reproducibility.
5. Folin-Ciocalteau reagent must be stored refrigerated, in amber coloured bottles (to protect it from light). A good quality reagent is straw yellow in colour. The reagent should not be used if it has a greenish tint. To such reagent add some bromine water and mix. The colour turns straw yellow and becomes usable.