Showing papers in "Methods in Enzymology in 1990"

Determination of carbonyl content in oxidatively modified proteins.

Abstract: Publisher Summary This chapter discusses methods to determine carbonyl content in oxidatively modified proteins. The methods described are (1) reduction of the carbonyl group to an alcohol with tritiated borohydride; (2) reaction of the carbonyl group with 2,4-dinitrophenylhydrazine to form the 2,4-dinitrophenylhydrazone; (3) reaction of the carbonyl with fluorescein thiosemicarbazide to form the thiosemicarbazone; and (4) reaction of the carbonyl group with fluorescein amine to form a Schiff base followed by reduction to the secondary amine with cyanoborohydride. Van Poelje and Snell have also quantitated protein-bound pyruvoyl groups through formation of a Schiff base with p-aminobenzoic acid followed by reduction with cyanoborohydride. Although a systematic investigation has not appeared, this method should also be useful in detecting other protein-bound carbonyl groups. Carbonyl content of proteins is expressed as moles carbonyl/mole subunit for purified proteins of known molecular weight. For extracts, the results may be given as nanomoles carbonyl/milligram protein. For a protein having a molecular weight of 50,000, a carbonyl content of 1 mol carbonyl/mol protein corresponds to 20 nmol carbonyl/mg proteins.

Role of free radicals and catalytic metal ions in human disease: an overview.

Abstract: Publisher Summary This chapter discusses the role of free radicals and catalytic metal ions in human disease. The importance of transition metal ions in mediating oxidant damage naturally leads to the question as to what forms of such ions might be available to catalyze radical reactions in vivo . The chapter discusses the metabolism of transition metals, such as iron and copper. It also discusses the chelation therapy that is an approach to site-specific antioxidant protection. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. A wide range of techniques is available to measure the rate of this process, but none is applicable to all circumstances. The two most popular are the measurement of diene conjugation and the thiobarbituric acid (TBA) test, but they are both subject to pitfalls, especially when applied to human samples. The chapter also discusses the essential principles of the peroxidation process. When discussing lipid peroxidation, it is essential to use clear terminology for the sequence of events involved; an imprecise use of terms such as initiation has caused considerable confusion in the literature. In a completely peroxide-free lipid system, first chain initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid refers to the attack of any species that has sufficient reactivity to abstract a hydrogen atom from a methylene group.

Malondialdehyde determination as index of lipid peroxidation

Abstract: Publisher Summary This chapter describes the malondialdehyde (MDA) as index of lipid peroxidation. The determination of malondialdehyde (MDA) has attracted widespread interest, because it appears to offer a facile means of assessing lipid peroxidation in biological materials. Malondialdehyde occurs in biological materials in free state and in various covalently bound forms. Urine also contains small amounts of MDA adducts with guanine, the phospholipid bases serine and ethanolamine, and other unidentified reactants. Free MDA is a minor and variable excretory product. It is apparent from the occurrence of these derivatives in urine that MDA forms adducts with proteins, nucleic acids, and other substances in vivo, and this compromises the assessment of lipid peroxidation in the tissues based on the determination of free MDA. The pH required for maximum yield of MDA varies among biological materials depending on the nature of the derivatives present. MDA may be generated during hydrolysis by the oxidation of polyunsaturated fatty acids (PUFA) in the sample and by the degradation of preexisting oxidation products. Pigments present in the sample, or generated during hydrolysis, also can interfere in the colorimetric assessment of MDA. These problems, and possibilities for their resolution, are discussed in the chapter.

Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal

Abstract: Publisher Summary This chapter discusses the methods used for the qualitative and quantitative determination of aldehydes in biological systems. It focuses on 4-hydroxynonenal (HNE) and malondialdehyde (MDA). 4-Hydroxynonenal is produced as a major product of the peroxidative decomposition of polyunsaturated fatty acids (PUFA) and possesses cytotoxic, hepatotoxic, mutagenic, and genoroxic properties. Increased levels of HNE are found in plasma and various organs under conditions of oxidative stress. In addition to HNE, lipid peroxidation generates many other aldehydes that may also be of toxicological significance. Malondialdehyde is in many instances the most abundant individual aldehyde resulting from lipid peroxidation, and its determination by thiobarbituric acid (TBA) is one of the most common assays in lipid peroxidation studies. In vitro MDA can alter proteins, DNA, RNA, and many other biomolecules. Recently, it has been demonstrated with monoclonal antibodies that malonaldehyde-altered protein occurs in atheroma of hyperlipidemic rabbits.

Rapid and Sensitive Sequence Comparison with FASTP and FASTA.

Abstract: The FASTA program can search the NBRF protein sequence library (2.5 million residues) in less than 20 min on an IBM-PC microcomputer and unambiguously detect proteins that shared a common ancestor billions of years in the past. FASTA is both fast and selective because it initially considers only amino acid identities. Its sensitivity is increased not only by using the PAM250 matrix to score and rescore regions with large numbers of identities but also by joining initial regions. The results of searches with FASTA compare favorably with results using NWS-based programs that are 100 times slower. FASTA is slightly less sensitive but considerably more selective. It is not clear that NWS-based programs would be more successful in finding distantly related members of the G-protein-coupled receptor family. The joining step by FASTA to calculate the initn score is especially useful for sequences that share regions of sequence similarity that are separated by variable-length loops. FASTP and FASTA were designed to identify protein sequences that have descended from a common ancestor, and they have proved very useful for this task. In many cases, a FASTA sequence search will result in a list of high scoring library sequences that are homologous to the query sequence, or the search will result in a list of sequences with similarity scores that cannot be distinguished from the bulk of the library. In either case, the question of whether there are sequences in the library that are clearly related to the query sequence has been answered unambiguously. Unfortunately, the results often will not be so clear-cut, and careful analysis of similarity scores, statistical significance, the actual aligned residues, and the biological context are required. In the course of analyzing the G-protein-coupled receptor family, several proteins were found that, because of a high initn score and a low init1 score that increased almost 2-fold with optimization, appeared to be members of this family which were not previously recognized. RDF2 analysis showed borderline z values, and only a careful examination of the sequence alignments that focused on the conserved residues provided convincing evidence that the high scores were fortuitous. As sequence comparison methods become more powerful by becoming more sensitive, they become more likely to mislead, and even greater care is required.

Flavonoids as antioxidants: determination of radical-scavenging efficiencies.

Abstract: Publisher Summary This chapter discusses the radical-scavenging efficiencies of flavonoids as antioxidants To test whether a certain substance acts as radical scavenger, the clearest evidence comes from the determination of reaction rate constants with a set of different specifically produced radicals In the tests discussed in the chapter, hydroxyl, azide, superoxide, linoleic acid peroxyl, tert-butoxyl, and sulfite radicals are used The spectral observation of radical reactions with flavonoids is assisted by the strong absorption characteristics of both the parent compounds and their respective aroxyl radicals Owing to the low solubility of flavonoids, conditions for pseudo-first order reactions with radical species can rarely be achieved for flavonoid aglycones Therefore, kinetic modeling calculations are employed The radical chemistry of flavonoids not only is of interest from a kinetic or mechanistic point of view but also offers considerable insight into structural relationships of highly evolved plant components First, the consistently high rate constants for attack by different types of radicals demonstrate the effective radicals scavenging capabilities of most flavonoids Second, owing to extensive electron delocalization as a prerequisite for radical stabilization, multiple mesomeric structures exist for aroxyl radical species of flavonoids

Quantitation of protein.

Abstract: Publisher Summary This chapter discusses various methods of estimating protein concentration. Absorption spectroscopy involves the absorption of a photon by an electron. Only those photons with a certain energy level can be absorbed as defined by the difference in energy between the orbital of the unexcited electron and a higher energy orbital. The peptide bond absorbs photons below 210 nm. Because of the large number of peptide bonds in a protein, this is a highly sensitive area of the protein spectrum. Although protein conformation and some absorption by tryptophan and tyrosine residues occurs in this region, less variability between proteins is observed than at 280 nm. There is a need to avoid storing buffers in plastic containers because some plastics leach plasticizers, which absorb at ultraviolet (UV) wavelengths. Detergents can also be troublesome because many absorb UV light. If the buffer or protein solution is cold, the outside of the cuvette may need to be wiped between each reading with a lint-free wiper and the readings should be made quickly after placing the cold solution into the cuvette, because atmospheric moisture may condense on the outside of the cuvette producing an erroneously high reading.

Avidin and streptavidin.

Abstract: Publisher Summary This chapter discusses the fundamental molecular properties of avidin and streptavidin. Carbohydrate-free avidin can be obtained by use of deglycosylating enzymes and it has been shown to be present in significant amounts in some commercial preparations from which it may be separated by use of lectin columns. Improvement on the original use of biotinyl cellulose came with the introduction of iminobiotinyl derivatives of Sepharose that utilized the pH dependence of the binding 24 to achieve efficient elution. In iminobiotin, the ureido group becomes a guanidinium group; the form in which this is uncharged is strongly bound. The gene for streptavidin has been cloned and sequenced with the ultimate objective of using it in general expression systems for detecting and isolating fusion proteins. The stability is greatly enhanced by biotin binding, because the total free energy of binding is about 330 kJ/mol of tetramer. The dissociation constant for biotin is so low that it can be estimated only from the ratio of the rate constants for binding and exchange. The binding is accompanied by a red shift of the tryptophan spectrum and by a decrease in fluorescence, either of which can be used as the basis for quantitative assays.

Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project.

Abstract: Publisher Summary There exists a sequence of 8 nucleotides, highly conserved at the boundary between an exon and an intron, referred to as the 5′ splice site (5′ ss). The boundary between an intron and an exon, the 3′ splice site (3′ ss), also exhibits a highly conserved sequence of 4 nucleotides, preceded by a pyrimidine-rich region. To interpret the deluge of sequence information generated by the human genome project, it is important to be able to identify genes in uncharacterized sequences. Without this capability, the sequence information is of little value. For each sequence in GenBank, the splice sites were located using the information in the annotation preceding the sequence, a FEATURES line being followed by multiple lines indicating different features present in the gene. The percentages of lariat windows with high-scoring branch point sequences in all GenBank categories, with the exception of viruses, are higher than those of random sequences. A notable observation is that even the window that contains the second highest frequency of branch points among the five windows analyzed always had a lower frequency of high scoring branch points than expected for a window with a random sequence.

Isolation of subcellular organelles.

Abstract: Publisher Summary This chapter explains the procedures for the isolation of nuclei, mitochondria, and lysosomes from cultured mammalian cell lines. Cultured Chinese hamster ovary (CHO) cells are routinely disrupted by low-pressure nitrogen cavitation. The cell pellets concentrate at the bottom of the bottles. All centrifugations are timed from when the rotor reaches running speed. The cavitated cells are homogenized with four strokes of a motor-driven pestle of a Potter–Elvehjem homogenizer. The motor drive is set so that the Teflon pestle almost stops turning as it reaches the bottom of the homogenizer. For the subsequent isolation of cytoplasmic organelles, nuclei and unbroken cells are rapidly separated from cytoplasmic organelles by differential sedimentation at low centrifugal force. Intact organelle membranes prevent substrate access to lumenal enzymes. Hence, organelle-specific enzyme activity is generally measured in the presence of nonionic detergent; comparison of enzyme activity in the presence and absence of detergent provides a quick method to assess organelle intactness.

Free radical initiators as source of water- or lipid-soluble peroxyl radicals.

Abstract: Publisher Summary This chapter discusses the role of free radical initiators as source of water- or lipid-soluble peroxyl radicals. Free radicals can be generated in either an aqueous or the lipid phase as required by using water-soluble 2,2′-Azo-bis(2-amidinopropane) dihydrochloride (AAPH) or lipid-soluble 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN). Admittedly, such azo compounds are not present in biological systems, but they are useful tools for studying quantitatively (1) the damage induced by free radicals on biological and related molecules and membranes and (2) the inhibition in model systems. The advantages are that the radicals can be generated at a constant rate at a specific site and that the rate of radical flux can be measured and controlled. Obviously, the most important characteristic of the free radical reaction is that it proceeds by a chain mechanism—that is, the rate of the overall reaction or the extent of damage can be quite significant even if the rate of initial radical formation or the amount of attacking radical is very small. It is, therefore, quite important to know how long the kinetic chain lasts. The chain length can never be known without knowing the rate of chain initiation or the radical flux. In fact, in the in vitro experiment, the kinetic chain length is as long as 100 in the oxidation of erythrocyte membranes induced by AAPH. Another advantage in using azo compounds is that, unlike peroxides, they are not explosive and can be handled easily and safely.

Electrospray ionization mass spectrometry.

Abstract: Publisher Summary This chapter discusses electrospray ionization mass spectrometry. Electrospray ionization (ESI) occurs during the electrostatic nebulization of a solution of charged analyte ions by a large electrostatic field gradient (approximately 3 kV/cm). Highly charged droplets are formed in a dry bath gas, at near atmospheric pressure. These charged droplets shrink as neutral solvent evaporates until the charge repulsion overcomes the cohesive droplet forces leading to a “Coulombic explosion.” Large oligopeptides and small proteins so far successfully examined by ESI-MS show a distribution of multiply charged molecular ions arising (in positive ion mode) by either proton or alkali ion attachment and no evidence of fragment ions because of fragmentation unless dissociation is induced during transport into mass spectrometer vacuum by collisions at higher energy. The largest protein so far examined is the native covalent dimer of bovine serum albumin with a M r of more than 130 kDa. It should be noted that in the case of noncovalently bonded species, such as protein subunits, the mass spectra show only the contributions of the individual subunits. These mass spectra show a distribution of charge states which usually spans about one-half the number of the most abundant charge state.

[9] Profile analysis

Abstract: Publisher Summary The profile method provides a convenient way to represent information about groups or families of sequences as well as a means to ask questions about the definition of protein families, the relationships between distantly related proteins, and the presence of sequence or structural motifs in proteins The observed positions of insertions and deletions in the sequences provide similar structural information This information is incorporated in the profile, and used to improve the detection of sequence patterns that represent structural motifs It is clear that certain three-dimensional structural motifs are shared by many proteins These structural motifs have patterns in their amino acid sequences that permit them to be recognized from the sequences alone Another use of profile analysis is based on profiles generated from sequences aligned using sequence information alone In this case, the profile can be considered to be specific for the protein family or super family as defined by sequence criteria A characteristic of profile analysis is that the score for aligning a residue at a given position varies depending on the observed conservation of residues at that position This may be contrasted with the approach of deriving a single consensus sequence for a family and using it, as a kind of family-specific probe, in standard alignment algorithms

Determination of superoxide dismutase activity by purely chemical system based on NAD(P)H oxidation.

Abstract: Publisher Summary This chapter discusses the superoxide dismutase (SOD) activity by purely chemical system based on nicotinamide adenine dinucleotide phosphate (NADPH) oxidation Most of the currently employed methods for the assay of SOD activity in tissue extracts are based on the ability of these enzymes to inhibit a superoxide-driven reaction The extent to which the rate of reaction is reduced can be taken as an indirect measurement of enzyme activity The generation of superoxide can be achieved by either enzymatic or nonenzymatic systems; subsequently, a suitable detection method, whether colorimetric, polarographic, or luminometric, according to different approaches must be devised Essential requirements for any assay are the sensitivity, reliability, and simplicity of the procedure so that it can be easily performed in the laboratory without the aid of expensive equipment and reagents The chapter discusses method that allows the determination of minute amounts of SODs, such as 2 ng, that are far below the detection limit of most employed methods Moreover, the usual detectors like nitro blue tetrazolium (NBT), cytochrome, or other chromogenic substrates, which are reduced by superoxide, might also be electron acceptors for reducing agents known to occur in biological samples This fact will explain the difficulties, often encountered with these assays, in attaining saturation levels and reproducible titration curves The method described in the chapter relies on the oxidation of NADPH, and this makes the detection less prone to interferences by aspecific reduction from cellular components

Introduction to avidin-biotin technology.

Abstract: Publisher Summary This chapter discusses the principles and advantages of the avidin–biotin system. The major distinguishing feature of the avidin–biotin system is the extraordinary affinity that characterizes the complex formed between the vitamin biotin and the egg-white protein avidin. The rationale in using the avidin–biotin system is based on the premise that if a compound biologically active with biotin is modified through its valeric acid side chain, the biological and physicochemical properties of the biotin-modified molecule are be changed significantly. If a reporter group of some sort is attached to the avidin molecule, the conjugate can be used for many different purposes. The avidin-conjugated probe can be added as a single chemically conjugated entity, or avidin can be applied in native form together with a biotinylated probe. The exceptionally high affinity and stability of the avidin–biotin complex ensures the desired conjunction of binder and probe. Biotin can readily be attached to most binders and probes; following biotinylation, the biological activity and physical characteristics are commonly retained.

Unified approach to alignment and phylogenies.

Abstract: Publisher Summary Two important considerations in the analysis of molecular sequences are alignment and phylogeny reconstruction. Typically, the procedure to find the phylogeny would presume an alignment of all sequences obtained manually. Each column would be treated independently and then be explained by a series of substitutions. Different branching configurations or tree topologies would be checked, and the one allowing for shortest total branch length would be chosen. The traditional dynamic programming algorithm can be generalized to compare sequence graphs and to find sequence subgraphs that are close to each other according to some sequence metric. Simultaneously, the subgraphs are aligned. The most informative distances for the tree construction process are calculated. When the method is applied to larger sequence numbers the complete distance matrix is not calculated because it is wasteful. Rearrangements are performed on the obtained tree to improve the overall fit of the tree to the distance data. The resulting tree is used to guide the alignment algorithm such that a parsimony tree is obtained that has the same topology as the distance tree. It is at this point that the graph comparison algorithm is used.

Predicting optimal and suboptimal secondary structure for RNA

Abstract: Publisher Summary There are two methods of predicting secondary structure: phylogeny and energy minimization. Phylogeny relies on alignment and subsequent folding of several sequences into similar structures for functionally analogous RNA. Energy minimization relies on thermodynamic parameters and computer. There are two versions of the program: LRNA folds linear sequences, and CRNA folds circular sequences. LRNA and CRNA have the same user interface. Information about the structure helps the program fold the sequence into a better secondary structure. The locations of single-stranded sites, double-stranded sites, or known helixes are easily entered in LRNA. In batch mode, a sequence of commands in a batch file is run non-interactively, usually in nonprime time. The user can type in the batch file using an editor or the interactive program BATGEN. BATGEN first asks for the batch file name and the header file name. The header file contains information placed at the top of the batch file, such as a system command to change directories.

Oxidative reactions of hemoglobin.

Abstract: Publisher Summary Hemoglobin readily undergoes one-electron oxidations and reductions, and it can act as a source or sink of free radicals An autoxidation of the heme groups produces O2– and, indirectly, H2O2 Hemoglobin also interacts with redox-active xenobiotics and metabolites, forming the xenobiotic radical and initiating a series of reactions that generate other radicals and oxidant species and often result in oxidative denaturation of the hemoglobin This chapter focuses on the measurement of oxidant-mediated changes to hemoglobin It defines the different oxidation products of hemoglobin and briefly describes general mechanisms for their production The oxidation of oxyhemoglobin (oxyHb)4 gives O2– and methemoglobin (metHb) If the globin structure is destabilized, metHb can convert to hemichrome in which either the distal histidine or an external ligand occupies the sixth coordination position of the ferric heme Hemichromes precipitate readily and are the main constituent of Heinz bodies Choleglobin is a term used to describe denatured hemoglobin in which the porphyrin ring has been hydroxylated or broken open Ferrylhemoglobin (ferrylHb or Hb2+H2O2) is an Fe(IV) complex formed from ferrous hemoglobin and H2O2 The reaction of metHb with H2O2 produces short-lived ferryl radicals, which are Fe (IV) species with the additional oxidizing equivalent localized on the globin

Progressive Alignment and Phylogenetic Tree Construction of Protein Sequences

Abstract: Publisher Summary The relationship of a set of related protein sequences can be expressed quantitatively in terms of a phylogenetic tree The accuracy of the tree naturally depends on the alignment of the sequences When there are more than two sequences, the problem lies in the gaps which must be introduced to align sequences optimally The preliminary set of pair wise measurements also reveals any sub clusters that may exist in the set These sub clusters can be treated as units during the alignment process, ensuring that the relative positions of the residues within the cluster will not be altered The matrix is then reduced by one, and all distance values related to this pair are averaged in a new matrix This procedure is repeated until the matrix is reduced to a single dimension The end result is a branching order with the associated branch lengths Aside from the fact that fewer programs are now involved, the single most important improvement is in the TREE program Formerly, to get the branch lengths of a phylogenetic tree, one had to manually construct a connectivity table, which is not only cumbersome but also error prone, especially when working with a large number of sequences

[22] Precipitation techniques

Abstract: Publisher Summary This chapter describes methods for the precipitation of proteins for preparative purposes. Proteins can be precipitated by causing perturbations in the solvent with respect to pH, ionic strength, and temperature. The art and science of protein fractionation by differential solubility and precipitability has reached a pinnacle, and a wide variety of precipitation methods have been discovered empirically. Although some adsorbents, such as alumina and calcium phosphate gels were used, most of the methods employed several steps of ammonium sulfate precipitation. Perturbations that can cause various conformational transitions include (1) a rise in temperature that can weaken the strength of dipolar interactions, such as hydrogen bonds and can favor formation of hydrophobic interactions and (2) a decrease in temperature that can cause the reverse. Quantitative aspects of the resultant transitions in structure depend on the total numbers of specific kinds of interactions and variation in energies among individual interactions of the same kind. The inclusion of water-miscible solvents in the medium in which the protein is dissolved represents a considerable perturbation.

Mass spectrometry of peptides and proteins by matrix-assisted ultraviolet laser desorption/ionization.

Abstract: Publisher Summary This chapter discusses mass spectrometry of peptides and proteins by matrix-assisted ultraviolet laser desorption/ionization. Three features make mass spectrometry an attractive analytical technique for biorganic molecules: very high sensitivity, potential for very highly accurate mass determination, and ability to elucidate structural information of the molecules under investigation. It is shown in this chapter that the goal of extremely high sensitivity has already been achieved in laser desorption/ionization (LDI-MS). It also yields important structural information—namely, on the level of the quaternary and, possibly, tertiary structures more than on that of the primary and secondary ones. The attainable accuracy in molecular mass determination still leaves much to be desired, but substantial improvements can be expected. Most lasers throughout the wavelength range from the far-ultraviolet to the far-infrared and with pulse durations from continuous wave down to the femtosecond (10 -15 sec) range have at some point been used to irradiate solid or liquid material in vacuum or under ambient pressure with the intent to couple energy from the laser beam into the sample via linear or nonlinear absorption and to transfer some of the sample material into the gas phase, a prerequisite for a mass spectrometric analysis.

Phospholipid hydroperoxide glutathione peroxidase.

Abstract: Publisher Summary This chapter discusses the phospholipid hydroperoxide glutathione peroxidase (PHGPX) activity in tissues and its purification. PHGPX is the second selenoenzyme discovered in mammals. Because PHGPX is a soluble enzyme has been purified from cell sap, it can be classified as a cytosolic enzyme. However, a substantial activity, which can be partially recovered by high ionic strength extraction, is present in membranes of subcellular organelles. Taking advantage of its peroxidation-inhibiting activity, PHGPX is discovered and purified in a simple lipid peroxidation test. The peroxidase activity, which is first identified on a partially purified preparation of the enzyme, is in fact measurable in crude fractions with some difficulties. Thereafter, the introduction, as peroxide substrate, of mixed micelles of phospholipid hydroperoxides and Triton X-100 greatly simplifies activity measurements. PHGPX activity, although measurable on liposomes or membranes containing hydroperoxides, is higher and linear when the substrate is in micellar form. To search for PHGPX activity in tissues, if activity is low and precise measurements are not possible using homogenates, the chapter uses two preparations, neither of them giving the true “total activity’ present in the tissue. In the first preparation, the tissue is broken in a Polytron homogenizer for 5 min in 3 volumes of 0.1 M Tris-HCl (pH 7.4), 0.3 M KCl, and centrifuged for 15 min at 15,000 g and 45 min at 100,000 g. In the second preparation, the tissue is homogenized in 3 volumes of 0.25 M sucrose, 20 mM Tris-HC1 (pH 7.4).

One-dimensional gel electrophoresis.

Abstract: Publisher Summary Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) is an excellent method to identify and monitor proteins during purification and to assess the homogeneity of purified fractions. The movements of the particles are retarded by interactions with the surrounding gel matrix, which acts as a molecular sieve. The opposing interactions of the electrical force and molecular sieving result in differential migration rates for the constituent proteins of a sample. SDS–PAGE overcomes the limitations of native polyacrylamide gel electrophoresis (PAGE) by imposing uniform hydrodynamic and charge characteristics on all the proteins in a sample mixture. During sample preparation, proteins are treated with hot sodium dodecyl sulfate (SDS). The anionic detergent binds tightly to most proteins at about 1.4 mg of SDS/mg of protein, imparting a negative charge to the resultant complexes. Most electrophoresis is done in vertical chambers in gel slabs formed between two glass plates. The slab format provides uniformity, so that different samples can be directly compared in the same gel.

Sequencing of peptides by tandem mass spectrometry and high-energy collision-induced dissociation.

Abstract: Publisher Summary This chapter discusses sequencing of peptides by tandem mass spectrometry and high-energy collision-induced dissociation. To ionize large polar molecules, such as peptides of a size encountered in the study of various aspects of protein structure, methods are used that impart little excess energy on the molecule. Furthermore, most of these "soft" ionization techniques produce the charged particle by addition or removal of a proton to form the very stable, even-electron ions (M + H) + or (M - H) - , that have little tendency to fragment. The first demonstration of the generation of large protonated peptides involved fast atom bombardment (FAB), but the data and methodology discussed in this chapter utilize either Xe ° or Cs + as primary particles. Peptides derived from proteins are linear molecules (at least after reduction of any disulfide bridges originally present) that consist of repeating units having an identical backbone, –NH–CH(R)–CO–, but differing in the nature of the side chain, R. There are only 20 different amino acids that occur in proteins (and are defined by the genetic code), in addition to the occasional occurrence of others formed by posttranslational processes.

Detergents: an overview.

Abstract: Publisher Summary Detergents are a class of compounds distinguished by their amphiphilic structure. Each molecule contains both hydrophilic and hydrophobic moieties, which give rise to the phenomenon of surface activity. Hydrocarbon chains, when singly dispersed in aqueous solution, are surrounded by structured, cagelike water. Aggregation of the chains releases some of this structured water, increasing the entropy and thereby decreasing the free energy of the system. The micellar structure adopted by a particular detergent depends to some extent on the structure of the monomer. Micellar structure also depends on experimental conditions, such as temperature, pressure, pH, ionic strength, and the presence of impurities. For example, many ionic detergents undergo a sphere-to-rod transition at high ionic strength. Some detergents exhibit a lower consolute temperature or cloud point. At the cloud point, a detergent solution passes from an isotropic micellar system to a two-phase system: One phase is depleted of detergent and the other is rich in giant micelles. The usually recorded cloud point is the temperature at which a warmed and cloudy 1% solution clears upon cooling.

Assessment of leukocyte involvement during ischemia and reperfusion of intestine.

Abstract: Publisher Summary This chapter discusses the role of neutrophilic polymorphonuclear leukocytes (neutrophils) in mediating the mucosal injury observed during ischemia and reperfusion of the small bowel. It describes methods used to quantify leukocyte infiltration into the postischemic intestinal mucosa. It also describes the methods used to determine whether leukocytes mediate the microvascular injury associated with reperfusion of the ischemic small bowel. Based on the results of studies on microvascular permeability and leukocyte kinetics in the postischemic bowel, the chapter also proposes a hypothesis to explain the relative roles of xanthine oxidase-derived oxidants and neutrophils in reperfusion-induced injury to the intestinal microvasculature. It proposes that during the ischemic period, adenosine triphosphate (ATP) is catabolized to yield hypoxanthine. The ischemic stress also triggers the conversion of nicotine adenine dinucleotide (NAD)-reducing xanthine dehydrogenase to the oxygen radical-producing xanthine oxidase. During reperfusion, molecular oxygen is reintroduced into the tissue where it reacts with hypoxanthine and xanthine oxidase to produce a burst of superoxide anion and hydrogen peroxide. In the presence of iron, superoxide anion and hydrogen peroxide react via the Haber–Weiss reaction to form hydroxyl radicals. This highly reactive and cytotoxic radical then initiate the lipid peroxidation of cell membrane components and the subsequent release of substances that attract, activate, and promote the adherence of granulocytes to microvascular endothelium. The adherent granulocytes then cause endothelial cell injury via the release of superoxide and various proteases.