Practical Biochemistry

Carbohydrates are one of the four major classes of biologically essential organic molecules in living organisms. They are the most abundant class of biomolecules in nature based on their mass. And they make up most of the organic bodies on earth due to their extensive role in every life form. Carbohydrates are polyhydroxy aldehydes or ketones or substances that result in such compounds upon hydrolysis. These macromolecules are composed of carbon (C), hydrogen (H), and oxygen (O). They are also referred to as saccharides (sakcharon = sugar or sweetness) since most of them have a sweet taste. Apart from that, carbohydrate serves as a primary energy source, a structural framework for nucleotides, and provides structural support to organisms. They also play a crucial role as mediators in cellular interaction. Carbohydrates are classified into monosaccharides, oligosaccharides, and polysaccharides. Monosaccharides are the simplest form of carbohydrates, and a few monosaccharides hydrolyse to form oligosaccharide, and many monosaccharides together form polysaccharides. They also act as a precursor for glycoproteins and glycolipids. In this section, we are discussing some basic concepts about carbohydrates. 

Qualitative tests for the determination of carbohydrates in the given sample are a crucial part of before getting into major analytical procedures. Every mono-, oligo-, and poly-saccharide vary depending upon the number of carbohydrate molecule present in it as well as changes in the side chains. Thus, every carbohydrate molecule has distinct functions and properties. Depending upon these physio-chemical properties of the carbohydrates, they respond to certain specific chemical reactions under certain conditions. Only mono- and dis-saccharides respond to the solubility test as they are soluble in water at room temperature. The Molisch test is only for the determination of the presence of carbohydrate, not depending on the types of it. The iodine test gives a result for polysaccharides. Whereas Fehling, Benedict and Osazone tests distinguish between reducing and non-reducing sugars. The Bradford test differentiates between mono- and di-saccharide-reducing sugars. Seliwanoff test is only for sucrose which is a non-reducing sugar. Bial’s test is to determine the presence of pentose sugars.

Quantification of carbohydrates by the anthrone method is a colourimetric assay. In this method, first, the complex carbohydrates are hydrolysed in a highly acidic medium, resulting in monosaccharides. Thereafter, these monosaccharides are dehydrated to form 5-hydroxymethylfurfural (5-HMF) or 2-furfural (2-F), followed by reacting with anthrone reagent. This reaction produces a blue-green complex which is colourimetrically determined. In DNSA method 3,5-dinitrosalicylic acid (DNSA), a yellow colour reagent reacts with reducing sugar’s carbonyl group and forms orangered colour compound 3-Amino-5-nitrosalicylic acid (ANSA). The quantity of carbohydrate present in a sample is determined by Beer-Lambert’s law. Apart from these techniques Folin-Wu, Hexokinase, Phenol-Sulphuric acid, Somogvi-Nelson, and GOPOD method are discussed in this chapter in detail.

Proteins are the ambidexterity macromolecules which play a crucial role in biological processes. Through proteins, genetic information is expressed. They exhibit enormous functions which are crucial in various pathways. Some proteins act as an enzyme which is vital for catalyzing various metabolic reactions in living cells. All proteins are made up of amino acids which join together via a peptide bond to form a polypeptide chain which is a precursor for the further development of protein. A nascent protein undergoes several post-translational modifications to achieve its true function. Based on the amino acids present in the protein, its function determines its role as hormones, enzymes, antibodies, transporters, etc. In this chapter, a brief introduction to protein’s structure and function is discussed.

Qualitative tests are the primary assays for detecting the presence of any protein in a given sample. Qualitative detection of proteins is based on the chemical interactions between amino acids and certain chemical reagents added to them. These chemical reactions yield colour formation, which corresponds to specific amino acid or a group of amino acids present in the sample. In this chapter, some of such most important assays for qualitative analysis are discussed in detail.

SDS-PAGE is the abbreviated form of sodium-dodecyl sulphatepolyacrylamide gel electrophoresis. It is a commonly used gel electrophoresis for the qualitative estimation of proteins. It is based on the principle that treatment of protein with SDS, an anionic detergent, causes a constant negative charge throughout the protein surface. It also breaks the protein chain into smaller peptide chains. Hence, the separation of proteins is solely based on the size and shape of the fragments generated and is independent of the charge of the protein. This, combined with polyacrylamide gel electrophoresis, allows for visualization of these fragments where the smaller fragments retain at the bottom of the gel. This method has been discussed in detail in this chapter.

Western blotting, also known as immunoblot, is a chromatography-based technique in which quantitative detection of proteins can be performed based on their molecular weight. This is often performed after the SDS-PAGE technique. It is used to identify, analyze and quantify proteins from a protein mixture. The major steps include 1. Separation of proteins (e.g., SDS-PAGE), 2. Transfer onto a solid support, 3. Utilizing primary and secondary antibodies for visualization. Western blotting can be used qualitatively and quantitatively. In this chapter, both of these techniques are explained in detail.

Quantitative tests for protein are essential for understanding the amount of protein present in a sample. Various tests have been developed, such as BCA, biuret, Bradford, and Lowry tests. These are the most common and standardised procedures for the determination of protein. The mentioned techniques are colourimetric assays. However, over a while, several more advanced techniques are also being developed. In this chapter, quantitative methods for protein are discussed. 

Protein purification is an essential step after protein isolation. Protein purification ensures the removal of all contaminants from the sample pool. Purification depends on the chemical and physical interactions of protein molecules and chemical moieties. These techniques are performed with tags and without tags. Tag purification involves ammonium sulfate precipitation and dialysis. Purification of proteins with tags utilizes affinity and size exclusion chromatography. There are various methods available for this, however, we will discuss some most common methods for protein purification.

 Nucleic acids are biomolecules which carry all the genetic information in the form of DNA and RNA. The nucleic acids are composed of primarily a 5-carbon sugar, a phosphate and a nitrogenous basis. These nitrogenous bases can be adenine, guanine, thymine, cytosine and uracil. Nucleic acids can be measured qualitatively by UV absorbance, diphenylamine method/orcinol method, fluorescence dyes and capillary electrophoresis. It can be measured qualitatively by agarose gel electrophoresis. 

Qualitative tests of nucleic acids are important to detect the presence or absence of nucleic acid in the given sample. It can be done by performing agarose gel electrophoresis. Nucleic acids are negatively charged and hence can be attracted towards a positive charge in an electric field. This phenomenon is used in agarose gel electrophoresis to detect nucleic acids. Based on the mass of nucleic acids, different bands are formed in the gel, which can be visualized using fluorescent dyes.

Nucleic acid quantification is essential in experiments associated with genetic engineering. Over periods, several techniques have been developed to quantify nucleic acids. UV-Vis spectrophotometer is the simplest technique where it is associated with absorbance, which is proportional to the concentration of the sample. There are some colourimetric methods, such as the diphenylamine (DPA) method, and the orcinol method, where nucleic acid reacts with diphenylamine and forms a bright blue colour complex, and orcinol reacts and forms a green colour complex. Apart from that, some advanced techniques, such as fluorescence techniques, are used for quantification. In this chapter, each of these methods is explained in detail.

Besides qualitative and quantification of nucleic acids, purification is an integral part of any genetic engineering experiments. In genetic engineering experiments, the purity of nucleic acid matters the most. Nucleic acid purification can be achieved by the phenol-chloroform method, alcohol-based purification, agarose gel electrophoresis, and chromatographic purification. These purifications are based on the interactions between bases of nucleic acids and chemical reagents. In this chapter, nucleic acid purification is explained in detail.

Lipids are water-insoluble hydrocarbon derivatives with many physiological functions. They are the structural components of membranes. The long chain fatty acid chains of lipids can be broken down to serve as energy fuels, as well as they play a role in intracellular signaling. The entire composition of lipid molecules in a cell constitutes its lipidome. In this chapter, we will discuss the different roles played by lipids by their chemical properties in detail. 

Qualitative tests are the primary assays for detecting the presence of any lipids in a given sample. Qualitative detection of lipids is based on the chemical interactions between components of lipids and certain chemical reagents added to them. These chemical reagents allow for the properties of lipids like hydrophobicity, translucency, or colourimetric analysis due to colour changes. These tests have been explained in detail in this chapter.

Quantification of lipids can be broadly classified into the following methods: gravimetric analysis, which employs the principle of phase separation, the acid value is based on the property of rancidity of fats, saponification value which relies on alkali required to saponify (hydrolyze) lipids containing mixture, iodine value depends on the interaction of fats with halogen iodine, and blood cholesterol estimation determines the cholesterol level by cholesterol esterase enzyme-based assay. These assays have been explained in detail in this chapter. Advanced techniques used for lipid quantification utilize the principles of chromatography (Thin-layer chromatography, gas chromatography, and HPLC) and spectroscopy (infrared spectroscopy, Raman spectroscopy, nuclear magnetic resistance spectroscopy, and fluorescence spectroscopy), and different colourimetric assays are discussed in detail in this chapter.

Different techniques are used in a biochemical laboratory to measure various parameters. For example, a pH meter is used to measure the pH of the sample and electrochemical cells are used to measure the redox potential. Zeta potential is another property which can be measured using zeta cells. Chromatography is a technique used to separate the components of a mixture. Two of the most common chromatography techniques are HPLC and ion exchange chromatography. HPLC is based on the interactions of the analytes in stationary as well as mobile phase. Whereas ion exchange chromatography is based on the interaction of ions and charged site of the stationary phase. 

A safety data sheet or SDS is a datasheet prepared by the manufacturer to illuminate the user about any potential hazard and preserved by the owner. SDS also provides information about the handling and working of the product. SDS is also known as a material safety data sheet (MSDS) or product safety data sheet (PSDS). It must include information about the physical, environmental health, and health hazards; safety precautions, and protective measurements; transportation and storing. Guidelines for the preparation of such SDS are laid down by the occupational safety and health administration (OSHA). This chapter briefly explains the SDS of the significant experimental hazards of those chemicals used in the previous chapters.