Methods in Enzymology 

Abstract: Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ2) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.

Abstract: Publisher Summary Catalase exerts a dual function: (1) decomposition of H 2 O 2 to give H 2 O and O 2 (catalytic activity) and (2) oxidation of H donors, for example, methanol, ethanol, formic acid, phenols, with the consumption of 1 mol of peroxide (peroxide activity) The kinetics of catalase does not obey the normal pattern Measurements of enzyme activity at substrate saturation or determination of the K s is therefore impossible In contrast to reactions proceeding at substrate saturation, the enzymic decomposition of H 2 O 2 is a first-order reaction, the rate of which is always proportional to the peroxide concentration present Consequently, to avoid a rapid decrease in the initial rate of the reaction, the assay must be carried out with relatively low concentrations of H 2 O 2 (about 001 M) This chapter discusses the catalytic activity of catalase The method of choice for biological material, however, is ultraviolet (UV) spectrophotometry Titrimetric methods are suitable for comparative studies For large series of measurements, there are either simple screening tests, which give a quick indication of the approximative catalase activity, or automated methods

Abstract: Publisher Summary This chapter discusses the sequencing end-labeled DNA with base-specific chemical cleavages. In the chemical DNA sequencing method, one end-labels the DNA, partially cleaves it at each of the four bases in four reactions, orders the products by size on a slab gel, and then reads the sequence from an autoradiogram by noting which base-specific agent cleaved at each successive nucleotide along the strand. This technique sequences the DNA made in and purified from cells. No enzymatic copying in vitro is required, and either single- or double-stranded DNA can be sequenced. Most chemical schemes that cleave at one or two of the four bases involve three consecutive steps: modification of a base, removal of the modified base from its sugar, and DNA strand scission at that sugar. Base-specific chemical cleavage is only one step in sequencing DNA. The chapter presents techniques for producing discrete DNA fragments, end-labeling DNA, segregating end-labeled fragments, extracting DNA from gels, and the protocols for partially cleaving it at specific bases using the chemical reactions. The chapter also discusses the electrophoresis of the chemical cleavage products on long-distance sequencing gels and a guide for troubleshooting problems in sequencing patterns.

Abstract: Publisher Summary This chapter discusses the analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Analyses of the Folin-Ciocalteu (FC) type are convenient, simple, and require only common equipment and have produced a large body of comparable data. Under proper conditions, the assay is inclusive of monophenols and gives predictable reactions with the types of phenols found in nature. Because different phenols react to different degrees, expression of the results as a single number—such as milligrams per liter gallic acid equivalence—is necessarily arbitrary. Because the reaction is independent, quantitative, and predictable, analysis of a mixture of phenols can be recalculated in terms of any other standard. The assay measures all compounds readily oxidizable under the reaction conditions and its very inclusiveness allows certain substances to also react that are either not phenols or seldom thought of as phenols (e.g., proteins). Judicious use of the assay—with consideration of potential interferences in particular samples and prior study if necessary—can lead to very informative results. Aggregate analysis of this type is an important supplement to and often more informative than reems of data difficult to summarize from various techniques, such as high-performance liquid chromatography (HPLC) that separate a large number of individual compounds .The predictable reaction of components in a mixture makes it possible to determine a single reactant by other means and to calculate its contribution to the total FC phenol content. Relative insensitivity of the FC analysis to many adsorbents and precipitants makes differential assay—before and after several different treatments—informative.

Abstract: Publisher Summary This chapter discusses microsomal lipid peroxidation Lipid peroxidation is a complex process known to occur in both plants and animals It involves the formation and propagation of lipid radicals, the uptake of oxygen, a rearrangement of the double bonds in unsaturated lipids, and the eventual destruction of membrane lipids, producing a variety of breakdown products, including alcohols, ketones, aldehydes, and ethers Biological membranes are often rich in unsaturated fatty acids and bathed in an oxygen-rich, metal-containing fluid Lipid peroxidation begins with the abstraction of a hydrogen atom from an unsaturated fatty acid, resulting in the formation of a lipid radical The formation of lipid endoperoxides in unsaturated fatty acids containing at least 3 methylene interrupted double bonds can lead to the formation of malondialdehyde as a breakdown product Nonenzymic peroxidation of microsomal membranes also occurs and is probably mediated in part by endogenous hemoproteins and transition metals The direct measurement of lipid hydroperoxides has an advantage over the thiobarbituric acid assay in that it permits a more accurate comparison of lipid peroxide levels in dissimilar lipid membranes