biochemical analysis techniques

M.Pharma (Quality Assurance)

Bio Evaluation Bio Evaluation Bio Evaluation Bio Evaluation

Biochemical analysis of Drugs

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Bio Evaluation Bio Evaluation Bio Evaluation Bio Evaluation TechniquesTechniquesTechniquesTechniques

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Submitted to- Me

(Asst.Prof ccp landr

Submiteed by-Shmmon Ahmad & Jassjeet Kharha

(M.Pharm Q.A)

DATEDATEDATEDATE----14/03/201314/03/201314/03/201314/03/2013

TechniquesTechniquesTechniquesTechniques


Metreyi Sharma

(Asst.Prof ccp landran)

Shmmon Ahmad & Jassjeet Kharha

(M.Pharm Q.A)


Biochemical analysis techniques of drugs

“Biochemical analysis techniques refer to a set of methods, assays, and procedures that enable(give a power) scientists to analyze the substances found in living organisms and the chemical reactions underlying life processes.”

To perform biochemical analysis of a biomolecule in a biological system, there is a need to design a strategy to detect the biomolecule & isolate it in pure form.

BIOMOLECULES:

A biomolecule is any molecule that is produced by a living organism, including large macromolecules such as proteins, polysaccharides, lipids, and nucleic acids, as well as small molecules such as primary metabolites and natural products.

Types of biomolecules

• Lipids,protein, polysaccharides, glycolipids, sterols, glycerolipids

• Vitamins

• Hormones, neurotransmitters

• Metabolites

• Monomers, oligomers and polymers

Most biomolecules occur in minute amounts in the cell, and for their detection and analysis it is required to purify them from any contamination. The methods for purification of biomolecules includes simple precipitation, centrifugation, and gel electrophoresis, chromatographic and affinity techniques.


BIOCHEMICAL ANALYSIS TECHNIQUES

• Spectrophotometery • Chromatography • Electrophoresis • Radioimmuno assay • Hyberidoma • ELISA • Centrifugation

Technician performing biochemical analysis

typically needs to design a strategy to detect that biomolecule, isolate it in pure form. from among thousands of molecules that can be found in an extracts from a biological sample, characterize it, and analyze its function. An assay, the biochemical test that characterizes a molecule, whether quantitative or semi-quantitative, is important to determine the presence and quantity of a biomolecule at each step of the study. Detection assays may range from the simple type of assays provided by spectrophotometric measurements and gel staining to determine the concentration and purity of proteins and nucleic acids, to long and tedious bioassays that may take days to perform.

The description and characterization of the molecular components of the cell succeeded in successive stages, each one related to the introduction of new technical tools adapted to the particular properties of the studied molecules. The first studied biomolecules were the small building blocks of larger and more complex macromolecules, the amino acids of proteins, the bases of nucleic acids and sugar monomers of complex carbohydrates.

The molecular characterization of these elementary components was carried out thanks to techniques used in organic chemistry and developed as early as the nineteenth century. Analysis and characterization of complex macromolecules proved more difficult, and the fundamental techniques in protein and nucleic acid and protein purification and sequencing were only established in the last four decades.

Most biomolecules occur in minute amounts in the cell, and their detection and analysis require the biochemist to first assume the major task of purifying them from any contamination. Purification procedures published in the specialist literature are almost as diverse as the diversity of biomolecules and are usually written in sufficient details that they can be reproduced in different laboratory with similar results. These procedures and protocols, which are reminiscent of recipes in cookbooks have had major influence on the progress of biomedical sciences and were very highly rated in scientific literature.


The methods available for purification of biomolecules range from simple precipitation, centrifugation, and gel electrophoresis to sophisticated chromatographic and affinity techniques that are constantly undergoing development and improvement. These diverse but interrelated methods are based on such properties as size and shape, net charge and bioproperties of the biomolecules studied.

Centrifugation procedures impose, through rapid spinning high centrifugal forces on biomolecules in solution, and cause their separations based on differences in weight. and refer to the process where biomolecules are separated because they adopt Electrophoresis techniques take advantage of both the size and charge of biomolecules different rates of migration toward positively (anode) or negatively (cathode) charged poles of an electric field. Gel electrophoresis methods are important steps in many separation and analysis techniques in the studies of DNA, proteins and lipids. Both western blotting techniques for the assay of proteins and southern and northern analysis of DNA rely on gel electrophoresis. The completion of DNA sequencing at the different human genome centers is also dependent on gel electrophoresis. A powerful modification of gel electrophoresis called twodimensional gel electrophoresis is predicted to play a very important role in the accomplishment of the proteome projects that have started in many laboratories.

Chromatography techniques are sensitive and effective in separating and concentrating minute components of a mixture and are widely used for quantitative and qualitative analysis in medicine, industrial processes, and other fields. The method consists of allowing a liquid or gaseous solution of the test mixture to flow through a tube or column packed with a finely divided solid material that may be coated with an active chemical group or an adsorbent liquid.

The different components of the mixture separate because they travel through the tube at different rates, depending on the interactions with the porous stationary material.

Various chromatographic separation strategies could be designed by modifying the chemical components and shape of the solid adsorbent material. Some chromatographic columns used in gel chromatography are packed with porous stationary material, such that the small molecules flowing through the column diffuse into the matrix and will be delayed, whereas larger molecules flow through the column more quickly.

Along with ultracentrifugation and gel electrophoresis, this is one of the methods used to determine the molecular weight of biomolecules. If the stationary material is charged, the chromatography column will allow separation of biomolecules


according to their charge, a process known as ion exchange chromatography. This process provides the highest resolution in the purification of native biomolecules and is valuable when both the purity and the activity of a molecule are of importance, as is the case in the preparation of all enzymes used in molecular biology.

The biological activity of biomolecules has itself been exploited to design a powerful separation method known as affinity chromatography. Most biomolecules of interest bind specifically and tightly to natural biological partners called ligands: enzymes bind substrates

and cofactors, hormones bind receptors, and specific immunoglobulinscalled antibodies can be made by the immune system that would in principle interact with any possible chemical component large enough to have a specific conformation. The solid material in an affinity chromatography column is coated with the ligand and only the biomolecule that specifically interact with this ligand will be retained while the rest of a mixture is washed away by excess solvent running through the column.

Once a pure biomolecule is obtained, it may be employed for a specific purpose such as an enzymatic reaction, used as a therapeutic agent, or in an industrial process. However, it is normal in a research laboratory that the biomolecule isolated is novel, isolated for the first time and, therefore, warrants full characterization in terms of structure and function. This is the most difficult part in a biochemical analysis of a novel biomolecule or a biochemical process, usually takes years to accomplish, and involves the collaboration of many research laboratories from different parts of the world.

Recent progress in biochemical analysis techniques has been dependant upon contributions from both chemistry and biology, especially molecular genetics and molecular biology, as well as engineering and information technology. Tagging of proteins and nucleic acids with chemicals, especially fluorescent dyes, has been crucial in helping to accomplish the sequencing of the human genome and other organisms, as well as the analysis of proteins by chromatography and mass spectrometry.

Biochemical research is undergoing a change in paradigm from analysis of the role of one or a few molecules at a time, to an approach aiming at the characterization and functional studies of many or even all biomolecules constituting a cell and eventually organs. One of the major challenges of the post-genome era is to assign functions to all of the gene products discovered through the genome and cDNA sequencing efforts.

The need for functional analysis of proteins has become especially eminent, and this has led to the renovated interest and major technical improvements in some protein separation and analysis techniques. Two-dimensional gel electrophoresis, high


performance liquid and capillary chromatography as well as mass spectrometry are proving very effective in separation and analysis of abundant change in highly expressed proteins. The newly developed hardware and software, and the use of automated systems that allow analysis of a huge number of samples simultaneously, is making it possible to analyze a large number of proteins in a shorter time and with higher accuracy. These approaches are making it possible to study global protein expression in cells and tissues, and will allow comparison of protein products from cells under varying conditions like differentiation and activation by various stimuli such as stress, hormones, or drugs.

A more specific assay to analyze protein function in vivo is to use expression systems designed to detect protein-protein and DNA-protein interactions such as the yeast and bacterial hybrid systems. Ligand-receptor interactions are also being studied by novel techniques using biosensors that are much faster than the conventional immunochemical and colorimetric analyzes.

The combination of large scale and automated analysis techniques, bioinformatic tools, and the power of genetic manipulations will enable scientists to eventually analyze processes of cell function to all depths.

BIOCHEMICAL ANALYSIS OF DRUGS:

Biochemical analysis of phospholipase D.

Phospholipase D (PLD) is distributed widely in nature, being present in various isoforms in bacteria, protozoa, fungi, plants, and animals. It catalyzes the hydrolysis of phospholipids, primarily phosphatidylcholine (PC), into phosphatidic acid (PA) and the head group, choline. It also catalyzes a transphosphatidylation reaction in which water is replaced by a primary alcohol to yield a phosphatidyl alcohol. This reaction is exclusive to PLD and is employed as a specific assay for the enzyme in in vivo systems. When the purified enzyme is assayed in vitro, the release of choline from PC can be utilized. This chapter describes production of a recombinant mammalian isozyme of PLD (PLD1) in baculovirus-infected insect cells and its purification. It also provides details of the assay procedure in the presence and absence of regulatory proteins in vitro. The assay of the enzyme in cells in vivo is also documented using labeling of endogenous PC by incubating the cells with (3)H-labeled fatty acid. Details of the assay utilizing the transphosphatidylation reaction are presented. In this, 1-butanol is employed as the primary alcohol and [(3)H]phosphatidylbutanol is isolated by thin-layer chromatography of lipid extracts from the cells. A variation of this assay is described using deuterated 1-butanol (1-butanol-d(10)) and detection of the synthesized deuterated phosphatidylbutanol species by mass spectrometry. Convenient alternative assays for PLD and diacylglycerol (DAG) lipase activity


based on fluorescence are also described. Many of the materials for these assays are available commercially, with the exception of the fluorescently labeled DAG substrate, which can be synthesized enzymatically in a simple one-step procedure

Biochemical analysis of proteins

In the field of protein and peptide biochemistry study, BioCentrum offers broad spectrum of electrophoretic analysis (SDS-PAGE, native electrophoresis, IEF, 2DE, WB) and chromatographic analysis (FPLC, HPLC). Moreover, we offer ability to determine the amino-acid composition of proteins and peptides, N-terminal sequence determination by Edman degradation, determination of secondary structure of proteins by circular dychroism (CD) and tertiary structure of proteins by X-Ray crystallography. Additionally, we offer analysis of protein-protein interaction by ELISA test and analysis of protein samples by MS.

� Electrophoretic pattern � Liquid chromatography services � Amino acid analysis � Sequencing of proteins and peptides � Analysis of CD spectra � ELISA tests

Amino acid analysis

Our laboratory offers amino acid analysis of proteins and peptides for determination of proteinaceus amino acids. Neither tryptophan nor cysteine is determined, no physiological nor modified amino acids are analyzed. The samples are hydrolyzed in gas-phase using 6M HCl during 24 h at 115 Celsius centigrades. Released amino acids are converted to phenylthiocarbamyl (PTC) derivatives and analysed on PicoTag 3.9x150 mm column (Waters) installed on a Waters HPLC unit. The detection limit is on a level of about 10 picomols but we suggest sending of a minimum 100 picomols of protein or peptide. We also accept proteins or peptides immobilized on a PVDF membranes. The samples for amino acid analysis should badvices presented at sequencing of liquid samples section.

The protein samples were hydrolyzed in gas phase using 6M HCl at 115 deg. C for 24 h. The liberated amino acids were converted into phenylthiocarbamyl (PTC) derivatives and analyzed by high-pressure liquid chromatography (HPLC) on a PicoTag 3.9x150 mm column (Waters, Milford, MA, USA)."


ELISA test ELISA is one of the most commonly used biochemical assay, applicable both in research and diagnostics. BioCentrum offers range of services in the field of ELISA optimization, including optimization of assay conditions such as blocking solution, time of incubation with antibodies and serum antibodies. Analyses are performed using fluorimetric and spectrophotometric detection.

Biochemical Analysis use UV/Visible spectroscopy for Bioresearch laboratories routinely use UV/Visible spectroscopy for the analysis of proteins, enzymes, nucleic acids and oligonucleotides. Whether a quick check of optical density, DNA-purity, enzyme analysis, or a quantitative determination of a protein or DNA, the Lambda series of UV/V is spectrometers can quickly and easily perform your analysis.

These reliable workhorse systems are controled by our powerful but easy-to-use UV WinLab™ Software. Proven in thousands of installations around the world, UV WinLab can scan a spectrum, collect wavelength programed data, work in concentration mode or collect time-drive data. It has a sophisticated report generator that takes advantage of stored report templates. The system can be operated from user created methods or from the bio specific methods supplied.

A large number of ready-to-run methods for routine biochemical analysis are included with our UV BioLab™ collection. With a single mouse-click the method is activated and the instrument is ready to measure your samples. Simply call-up the method and start it. The UV BioLab collection of pre-programmed methods is available at no extra charge for all Lambda systems, and include five key method groups:

• Nucleic acid analysis

• Protein analysis

• Kinetic analysis

• General quantitative analysis

• General UV/Vis spectroscopic analysis

Quick and Easy Protein Analysis

Protein analysis and most common colorimetric protein assays can be done quickly and easily with the pre-programmed methods included with our UV BioLab


collection. Simply prepare the standard solutions and samples according to protocol, and activate the respective method. The measurements will automatically be taken with one simple click of the mouse. Oligocalculator results for the concentration and Tm of a 25mer oligonucleotide together with its Molecular Mass and Molar Extinction Coefficient.

The result page of DNA purity (Ratio A260/A280) and concentration determination.The following protein methods are included with the UV BioLab collection. If sample automation is desired, a sipper system may be added. In addition to the provided method, developers may choose to create their own methods with UV WinLab software.

• OD280 for direct protein determination

• Lowry protein method for high and low concentration range

• Dye binding protein assay according to Bradford (see Figure 4, Coomassie blue)

• Biuret method for protein Quantification

• BCA assay

• Warburg/Christian method for

direct protein determination at 280/260 nm. Automated Kinetic Experiments With UV WinLab’s integrated UV KinLab™ module and pre-programmed methods, it is easy to monitor enzyme reactions in order to determine enzyme activity.

Absorbance versus time is displayed on-line, and enzyme activity calculated from the resulting slope of the reaction curve. This can be done automatically with a defined time interval, or calculated post-run with experiment specific timing Automation of kinetic measurements in UV/Vis spectrometry is usually achieved by use of manual or automated cell changers. Enzyme tests are often time-consuming with a typical test lasting between 3 and 15 minutes. Automated thermostatted Cell Changers can help reduce the measurement time for multiple samples.

Cell Changer Systems for Any ApplicationOur 8+1 and 9+1 Cell Changer systems significantly increase sample throughput and are optimized for time-dependent UV/Vis spectroscopic measurements like enzyme kinetics, they may however be used for all basic methods:

• Timedrive and Kinetics for single wavelength measurement.


• Wavelength program for measurement at up to 8 different wavelengths including result calculation.

BIOCHEMICAL ANALYSIS OF HUMAN DNA2 ATPase assay

Purified hDna2 was incubated in 20 µl of reaction buffer containing 40 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 25 mM NaCl, 2.5 mM DTT, 0.1 mg/ml BSA, 5% glycerol (v/v), 1 µg of oligonucleotide (primer, 22 bases) and various concentrations of [γ-32P]ATP at 37°C. These conditions were determined as optimal in separate titrations of ATP, NaCl and time. The reaction was stopped by adding EDTA to a final concentration of 4 mM, and the reaction mix (0.5 µl) was spotted onto a polyethyleneimine cellulose plate (SELECTO SCIENTIFIC), which was then developed in 0.5 M LiCl, 1 M formic acid solution. The results were analyzed using the STORM PhosphoImager.

Nuclease assay

In general, nuclease activities of hDna2 were measured using a standard reaction mixture (20 µl) containing 50 mM Tris–HCl, pH 7.5, 25 mM NaCl, 2 mM DTT, 0.25 mg/ml BSA, the 5′- or 3′-32P-labeled DNA substrate, and various concentrations of MgCl2 and ATP as indicated in the figure legends. NaCl inhibited the nuclease activity (20 times inhibition at 125 mM) but 25 mM NaCl was minimally inhibitory and included in the reactions to stabilize the oligonucleotide substrates. After incubation at 37°C for 15 min, reactions were stopped with 2× denaturing termination dye (95% deionized formamide, 10 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol), and boiled for 5 min. The cleavage products were separated on a 12% sequencing gel (SequaGel, National Diagnostics) using Model S2 electrophoresis apparatus (BRL, 39 cm plate) and analyzed using the PhosphoImager. Products were quantified using the ImageQuant software on the phosphorimager, Substrate cleaved (%) is calculated as follows: Substrate cleaved (%) = (product bands)/(substrate bands + product bands) × 100.

Helicase assay

Helicase assays were performed with the nuclease-deficient mutant of hDna2 (D294A). The standard reaction mixtures contained 50 mM Tris–HCl, pH 7.5, 25 mM NaCl, 2 mM DTT, 0.25 mg/ml BSA, 4 mM MgCl2, 4 mM ATP and 32P-labeled helicase substrate. After incubation at 37°C for 1 h, reactions were stopped with 5× stop solution (60 mM EDTA, 40% sucrose, 0.6% SDS, 0.25% bromophenol blue and 0.25% xylene cyanole FF). Reaction products were then


separated using 8% native polyacrylamide gels containing 0.1% SDS, and detected with PhosphoImager.

hDna2 nuclease substrates

All oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). Oligonucleotide sequences are listed in Table 1. The locations of biotinylation are indicated as underlined nucleotides in the table. The 5′ and 3′ end labeling of oligonucleotides were performed as described previously (33). Oligonucleotides were annealed as described in the figure legends to form various structures. Flap substrate for the 5′–3′ nuclease assay were made by annealing a downstream oligonucleotide, template, and upstream oligonucleotide at molar ratio of 1:2:4. The upstream oligonucleotide was omitted to make the forked substrate.

biochemical analysis techniques

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