Preface   Biochemistry is the science which deals with the chemical composition, synthesis, degradation and interaction of compounds found in living organisms. More simply it encompasses understanding of life processes in terms of chemical phenomena. The theme of biochemistry is to correlate the molecular structures with biological functions. Today, the growth of all other biological sciences such as biotechnology, biology, physiology, genetics, pathology, microbiology, medicine, agriculture etc. is dependent upon biochemistry. Biochemistry has an important role to play in all the life processes and technological developments in the area of food energy public health and pollution control in order to meet the requirement with the rapidly increasing world population in the years to come. Biochemistry is an experimental science. Rapid advances in our knowledge of life processes have been made because of the development of newer laboratory techniques and appropriate methodologies which yield satisfactory results. Although there are quite a number of books available on general and clinical biochemistry, the laboratory techniques and biochemical methodologies are found scattered in various journals and others. Hence, it is laborious and time consuming for the students and scientists so as to search for a desirable method of analysis. Obviously, there is a need for a book which at one place could provide the various biochemical methods of analysis that are in frequent use in research and class works. This practical laboratory manual entitled "Biochemistry : Methods and Techniques" has been written to help researchers, teachers and students for proper understanding and use of the major techniques and methods employed in biochemistry. The book is primarily intended for the students, scholars and scientists engaged in agricultural research, working in areas like plant biochemistry, plant breeding, plant biotechnology, genetics, plant physiology, plant pathology, agricultural microbiology, soil science, seed science, agricultural chemistry, food sciences, agricultural entomology and other related sciences. However, this manual is designed to meet the requirements of teachers, researchers, and students alike who are engaged in the study of biochemistry. We feel extremely happy to bring out this practical guide on experimentation in the field of biochemistry. It is quite certain that it would serve the purpose of being useful to students as a dependable laboratory manual. This book has been prepared on course basis inclusive of some advanced techniques that we have standardized. The chemical composition of different media and their preparations are presented in the appendices. There are 88 exercises in this manual. The exercise contains a number of illustrations that complement the text. It is hoped that the manual would not only be educative but would also serve an as ready reckoner for readers intrigued by this subject. Besides the above 88 exercises some definitions; weights and measures; preparation of buffers; standard solutions; indicators; stains etc. and other relevant information have been inpregnated in the appendices. In the end we owe the responsibility for mistakes, omissions etc, if any and would be highly thankful to those bringing out the same to our notice along with the valuable suggestions for further improvement of this laboratory manual.

Hypothesis become theories and theories attain rank of laws after withstanding rigorous experimental tests. Feasibility of a process is confirmed in the laboratory. Qualitative and quantitative analyses give complete chemical picture of the substance. It is with these considerations in mind we proceed to learn what is there in a laboratory.

Carbohydrates are synthesized from CO2 and H2O in chlorophyll containing plants during photosynthesis. The term carbohydrate was originally coined for this class of compounds as most of them were hydrates of carbon or could be represented by the general formula Cn(H2O)n. Not all the carbohydrates however, have this empirical formula, for example deoxyribose (C5H10O4) and glucosamine (C6H13O5N). Further lactic acid (C3H6O3) formaldehyde (HCHO or CH2O) and acetic acid (CH3­.COOH or C2H4O2) with this general formula is not a carbohydrate. Carbohydrates are defined as the compounds having either an aldehyde or a ketone group or a modified aldehyde or ketone group and other carbon atoms with alcoholic (-OH) group. Hence the continued usage of the term carbohydrate is for convenience rather than exactness.

Amino acids are essential components of all living cells as building blocks of proteins. They are also metabolically active and supply substrates for many other biochemical reactions. In a bacterial cell, about 0.4% of the dry weight is amino acids. The amino units in other living systems varies according to species, age and part of the body.

Lipids are a hetrogenous group of substances, widely distributed throughout the plant and animal kingdom. In plants they are present in the seeds nuts and fruits, while in the animals they are stored in adipose tissues, bone marrow and nervous tissues. Lipids are relatively insoluble in water and readily soluble in organic solvents such as ether, chloroform, carbon disulfide, benzene, hot alcohol etc. The term lipid was used by the German biochemist Bloor in 1943 for a major class of tissue components and foodstuffs.

Nucleic acids like proteins occur in all living cells. They device their name because of their primary occurrence in the nucleus and acidic nature. Deoxyribose nucleic acid (DNA), one of the two nucleic acids is a major component of chromosomes. Small amounts of DNA are also found associated with chloroplasts and mitochondria.

The term enzyme was coined in 1878 by Friedrich Wilhelm Kuhne to designate these biological catalysts that had previou-sly been called ferments. As they quicken most of the chemical reactions occurr-ing in the body, the enzymes have been designated as the “manifestations of nature’s impatience”.

Franz Holfmeister’s (LT, 1850-1922) definition, “vitamins are substances which are indispensable for the growth and maintenance of the animal organism, which occur both in animal and plants and are present in only small amounts in food”, still holds good, although it has been interpreted in various ways.

The pigments are energy-rich organic compounds. The potential chemical energy of these compounds comes from the light energy. The light enrgy to be effective in photosynthesis must be absorbed by a suitable pigment. This vital role is performed by a suitable pigment. This vital role is performed by the green pigment. Chlorophyll, carotenoid (carotenes and xanthophylls), anthocynin and phycibilins.

Phenolics are broadly distributed in the plant kingdom and are the most abundant secondary metabolites of plants. Plant polyphenols have drawn increasing attention due to their potent antioxidant properties and their marked effects in the prevention of various oxidative stress associated diseases such as cancer. Polyphenols are a structural class of mainly natural, but also synthetic, and semisynthetic, organic chemicals characterized by the presence of large multiples of phenol structural units. The number and characteristics of these phenol structural underlie the unique physical, chemical, and biological (metabolic, toxic, therapeutic, etc.) properties of particular members of the class. The term polyphenol derives from poly-, from the ancient Greek word (polus, meaning “many, much”) and the word phenol which refers to a chemical structure/substructure formed by attaching to an aromatic phenyl or benzenoid ring an alcohol-type hydroxyl (-OH) group (giving rise to the “-ol” suffix). The term “polyphenol” has been in use since 1894.

An anti-nutritional factor is a substance which, when present in human or animal foods, reduces growth. Examples are phytate, protease inhibitors (notably soybean trypsin inhibitor) and excessive dietary fiber. Anti-Nutritional Factors     ·    Protease inhibitors inhibit the activity of trypsin, chemotropism and other proteases. They are found in legumes such as beans and peas, but also in cereals, potatoes, and other products. Their presence results in impaired growth and poor food utilization.

Thiamann (1948) designated the plant hormones by the term ‘phytohormones’ in order to distinguish them from animal hormones. He defined a phytohormone as “an organic compound produced naturally in higher plants, controlling growth or other phy-siological functions at a site remote from its place of production and active in minute amounts.” According to Johannes van Over-beek (1950), the plant hormones are defined as “organic compounds which regulate plant physiological process – regardless of whether these compounds are naturally occurring and/or synthetic; simulating and/or inhibitory; local activators or substances which act at a distance from the place where they are formed.”

Appendices Appendix – I Definitions 1.Atomic weight: Atomic weight of an element is the relative weight of the atom on the basis of oxygen as 16. For example, Atomic weight of sodium is 23. 2.Molecular weight: The sum of the atomic weights of all the atoms in a molecule is its molecular weight. For example, Molecular weight of H2SO4 is 98, since- 3.Equivalent weight: Equivalent weight of a substance is the number of grams of the substance required to react with, replace or furnish one mole of H2O+ or OH-. The equivalent weight of an acid is the weight that contains one atomic weight of acidic hydrogen i.e., the hydrogen that reacts during neutralization of acid with base. For example, the equivalent weight of H2SO4 is 49. Since H2SO4 contains two replaceable hydrogens, equivalent weight is molecular weight/2 i.e., 98/2=49. 4.Percent Solution (w/v): One percent solution of a substance contains one gram of the substance in 100 mL of the solvent. If v/v is given, it means 1mL in 100 mL of solvent. 5.Molar solution (M): One molar solution of a substance contains one mole or one gram molecular weight of the substance in one litre of solution. For example, 1 M NaOH contains 40g sodium hydroxide in one litre solution. Likewise, one millimolar solution of a substance contains 6.Normal solution (N): One normal solution of a substance contains on equivalent or one gram equivalent weight of the substance in one litre solution (i.e., molecular weight divided by the hydrogen equivalent of the substance).