Good Laboratory Practice (GLP)

What exactly the GLP is? GLP is often confused with the standards of safe laboratory practices such as wearing aprons, gloves, and safety masks. GLP goes beyond the safe laboratory practices. An internationally recognized definition of GLP goes like this: Good Laboratory Practice (GLP) embodies a set of principles that provides a framework within which laboratory studies are planned, performed, monitored, recorded, reported, and archived . New Zealand and Denmark were the first to introduce GLP in1972. United States was the next to introduce GLP in response to the poor scientific practices prevalent in US around that time. In early 1970s, US Food and Drug Administration realized the poor laboratory practices throughout United States. FDA also became aware of the fraudulent data produced by several toxicology laboratories; Industrial BioTest Labs being the most noted one. An international economic organization, the Organisation for Economic Co-operation and Development (OECD) published the principles of GLP in 1981 under the name ‘OECD Guidelines for the Testing of Chemicals' that were internationally accepted. These guidelines are available on the OECD website: http://www.oecd.org/env/ehs/testing/oecdguidelinesforthetestingofchemicals.htm. GLP makes sure that the raw data generated during the study are traceable thereby ensuring the authenticity of the published data. GLP mandates a Quality Assurance unit that is required for monitoring and auditing the studies that are underway.

Majority of the experiments discussed in this course will, directly or indirectly, utilize two major classes of biomolecules, proteins and nucleic acids. It is therefore worth reviewing the chemical nature and the structure of the proteins and nucleic acids.

Chemical and structural features of proteins and nucleic acids

Amino acids and Proteins

Proteins constitute the cellular machinery that carry out majority of the biological reactions. They are linear polymers of L-α-amino acids. The structural of a typical L-α-amino acid is shown in figure 1.2. The R group, also known as the side-chain of an amino acid, makes a repertoire of 20 different amino acids. In proteins, amino acids are linked together through amide bonds formed between α-amino group and the α-carboxylate group as shown in figure 1.2B. As both α-amino and α-carboxylate groups are involved in making the peptide bond, the chemistry of the polypeptide is determined by the chemistry of the side-chains of the constituent amino acids. Based on the chemistry of their side-chains, amino acids have been classified as polar and non-polar. Polar amino acids have been further classified into neutral, acidic, and basic amino acids depending on the charge on the side-chain at neutral pH. Lysine side-chain, for example, has a terminal amino group that is protonated at neutral pH; lysine, therefore, is identified as a basic amino acid. Aspartic acid and glutamic acid have carboxyl group in their side chains; at neutral pH, the side chains are ionized making them the acidic amino acids.

The amino acids in a protein are linked through an amide bond, called a peptide bond (Figure 1.2B). Delocalization of the nitrogen's unshared pair of electrons over carbonyl group imparts a partial double bond character to the peptide bond. This implies that rotation about the C(O)–N(H) is not allowed, putting constraints on the conformations a polypeptide backbone can adopt. The rotations about C_α –C(O) and N–C_α, however, are allowed; and these dihedral angles are referred to as psi (ψ) and phi (φ), respectively (Figure 1.2B). The amino acid sequence of a protein, which is also termed as its primary structure, is read from N-terminal to C-terminal. The polypeptide regions within a protein can adopt local ordered structures (very similar φ and ψ values for a continuous stretch of amino acids) called secondary structures (Figure 1.2C); α-helices and β-sheets are two such secondary structures. Further assembly of the polypeptide chain leads to a compact structure, termed the tertiaty structure (Figure 1.2D). Many proteins function as multimers i.e. they have more than one polypeptide chains in their functional form that interact with each other through non-covalent interactions. Such multimeric protein structures are referred to as the quaternary structures (Figure 1.2D).

Figure 1.2 Structures of amino acids and proteins. Panel A represents the structure of a typical amino acid; the side-chain R determines the chemistry of the amino acid in a protein. Panel B shows the resonance structures and the partial double bond character of peptide bond. Panel C defines the primary and secondary structures of the proteins. Panel D shows the definitions of tertiary and quaternary structures.