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1.1. INTRODUCTION The objective of many bioassay methods is to selectively quantitate a single biomolecule, such as a particular enzyme or antibody, or to determine the presence or absence of a known DNA sequence in an unknown sample. Methods for these very selective assays will be considered in later chapters. When faced with a true unknown, for example, during the isolation or puri cation of a biomolecule, it can be important to characterize the unknown matrix, or the components in the unknown solution that are present along with the species of interest. This involves the estimation of the total quantity of the different types of biomolecules. Biochemists often estimate the total quantity of protein and nucleic acid in an unknown by the nomograph method.1 In this method, the absorbance of the unknown solution in a 1-cm cuvette is measured at 260 and 280 nm. The nomograph (Fig. 1.1) is then used to estimate concentrations. The nomograph method is rapid and involves no chemical derivatization, but suffers from several disadvantages. Interferences result from any species present in the unknown, other than protein or nucleic acid, that absorb at these wavelengths. Phenol, for example, which is used at high concentrations during the puri cation of nucleic acid, absorbs at both 260 and 280 nm and leads to overestimation of total nucleic acid. Furthermore, the nomograph method does not distinguish between deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and is useful only for samples containing relatively high concentrations of protein and nucleic acid. In this chapter, we will consider simple colorimetric and uorometric methods for the quantitation of total protein, DNA, RNA, carbohydrate, and free fatty acid. All of the methods are based on the generation of a chromophore or uorophore by a selective chemical reaction.
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Bianalytical Chemistry, by Susan R. Mikkelsen and Eduardo Corton ISBN 0-471-54447-7 Copyright # 2004 John Wiley & Sons, Inc.
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SPECTROSCOPIC METHODS FOR MATRIX CHARACTERIZATION
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Figure 1.1. Nomograph used to estimate total protein and nucleic acid concentrations. [Reprinted, with permission, from Calbiochem, San Diego, CA.]
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1.2. TOTAL PROTEIN The three most common assays for total protein2 are the Lowry (enhanced copper), Smith (bicinchoninic acid, BCA), and Bradford (Coomassie Blue) methods. All are colorimetric methods, and are based on the generation of absorbing species in proportion to the quantity of protein present in the sample. The ninhydrin assay is a recently reported promising method.
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1.2.1. Lowry Method The Lowry, or enhanced alkaline copper method, begins with the addition of an alkaline solution of Cu2 to the sample. The copper forms a complex with nitrogen atoms in the peptide bonds of proteins under these conditions, and is reduced to Cu . The Cu , along with the R groups of tyrosine, tryptophan, and cysteine residues of the protein then react with the added Folin Ciocalteau reagent, which contains sodium tungstate, sodium molybdate, phosphoric acid, and HCl (W6 /Mo6 ). During this reaction, the Cu is oxidized to Cu3 , which then reacts with the pep CO Co tide backbone to produce imino peptide (R1 N C(R2) R3) plus Cu2 , and the Folin reagent is reduced to become molybdenum tungsten blue. Absorbance is measured in glass or polystyrene cuvettes at 720 nm, or if this value is too high (>2), at 500 nm. A calibration curve obtained with standard protein solutions [e.g., bovine serum albumin (BSA)] is used to obtain total protein in the unknown. Under optimum conditions, and in the absence of reactive side chains, it has been shown that two electrons are transferred per tetrapeptide unit; however, proteins, with signi cant proline or hydroxyproline content, or with side chains that can complex copper (such as glutamate) yield less color. The side chains of cysteine, tyrosine and tryptophan contribute one, four and four electrons, respectively.3 Note that different proteins will produce different color intensities, primarily as a result of different tyrosine and tryptophan contents. With reagents prepared in advance, the Lowry assay requires $ 1 h. A 400-mL sample is required, containing 2 100-mg protein (5 250 mg/mL). Nonlinear calibration curves are obtained, due to decomposition of the Folin reagent at alkaline pH following addition to the sample that results in incomplete reaction. Interferences include agents that acidify the solution, chelate copper, or cause reduction of copper(II). 1.2.2. Smith (BCA) Method The Smith total protein assay is also based on the initial complexation of copper (II) with peptides under alkaline conditions, with reduction to copper(I). The ligand BCA is then added in excess, and the purple color (562-nm peak absorbance) develops upon 2:1 binding of BCA with Cu (Fig. 1.2).
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