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Showing papers in "Methods in Enzymology in 1967"


Book ChapterDOI
TL;DR: This chapter discusses the mitochondrial respiratory control and the polarographic measurement of ADP : O ratios and the principle of the oxygen electrode has been summarized, and the design of the vibrating oxygen electrode for use with speetrophotometric studies is illustrated.
Abstract: Publisher Summary This chapter discusses the mitochondrial respiratory control and the polarographic measurement of ADP : O ratios. The polarographic oxygen electrode technique is used for measuring rapid changes in the rate of oxygen utilization by cellular and subcellular systems. Although the polarographic method measures changes in oxygen concentration of photosynthetic systems, yeast cells, and nerve, but the oxygen electrode technique is applied to a study the mitochondrial respiration and oxidative phosphorytation. The principle of the oxygen electrode has been summarized, and the design of the vibrating oxygen electrode for use with speetrophotometric studies is illustrated. The oxygen electrode apparatus can be calibrated in a number of ways. A more accurate calibration of oxygen content can be obtained by gas equilibration with various nitrogen-oxygen mixtures. When tightly coupled mitochondria are suspended in an isotonic buffer, a slow rate of oxygen uptake is measured in the presence of substrate and absence of ADP. Addition of ADP causes an immediate increase in the rate of oxygen utilization. The concentration of oxygen utilized is proportional to the amount of ADP phosphorylated to ATP. The type of oxygen electrode tracings is presented from which an ADP : O ratio (equivalent to a P : O ratio) can be directly calculated.

1,810 citations


Book ChapterDOI
TL;DR: This chapter discusses the cytochrome c oxidase from beef heart mitochondria, which is inhibited by cyanide, azide, hydroxylamine, and sodium sulfide.
Abstract: Publisher Summary This chapter discusses the cytochrome oxidase from beef heart mitochondria. Cytochrome c oxidase is mostly assayed by the spectrophotometric method. The rate of oxidation of ferrocytochrome c is measured by following the decrease in the absorbency of its α-band at 550 mμ. The activity of cytochrome c oxidase may be defined in terms of the first-order velocity constant. The oxidation-reduction components of isolated cytochrome c oxidase are cytochrome a, cytochrome a 3 , and copper. Cytochrome c oxidase can also be assayed by measuring oxygen uptake either manometrically or polarographically. Two procedures for purifying cytochrome c oxidase from beef heart mitochondria are described. The enzyme obtained by procedure I is less pure based on its activity and spectrum. Preparations of cytochrome oxidase are stored best in 0.25M sucrose (pH 7.0-7.5) at –15° at a protein concentration of 1 mg/ml. The composition of cytochrome oxidase prepared by procedure II is tabulated. Ferrocytochrome c donates electrons directly to cytochrome oxidase. The reaction as measured spectrophotometrically obeys first-order reaction kinetics. Cytochrome oxidase is inhibited by cyanide, azide, hydroxylamine, and sodium sulfide.

1,244 citations


Book ChapterDOI
TL;DR: This chapter describes the isolation of liver or kidney mitochondria and the method for testing the quality of mitochondria, with the exception that the mitochondrial pellet need be washed only once.
Abstract: Publisher Summary This chapter describes the isolation of liver or kidney mitochondria. The selected tissue is disrupted by homogenization in cold isotonic sucrose. Differential centrifugation is then employed to separate the mitochondria from cell debris, red blood cells, nuclei, microsomes, and soluble components. For the isolation of liver mitochondria, the homogenate is distributed into Lusteroid centrifuge cups and centrifuged at 600 g for 10 minutes. The supernatant fraction is decanted and saved. The pellets may be dispersed by using the side of a stirring rod against the wall of the cup or by handoperating the homogenizer. The resuspended material is centrifuged at 600 g for 10 minutes. The supernatant fractions are combined. The pellets are discarded. This washing contributes not only to the yield of the final mitochondrial preparation, but also to its integrity, apparently by permitting the recovery of the larger mitochondria. For the isolation of kidney, the kidney capsule is removed by gently squeezing the kidney through the thumb and forefinger. The kidney is then cut sagittally. The medullary portion is removed and discarded. Mitochondria are then prepared from the cortex following the method described for liver, with the exception that the mitochondrial pellet need be washed only once. Method for testing the quality of mitochondria is also discusses in the chapter.

1,077 citations


Book ChapterDOI
TL;DR: This chapter discusses the determination of cystine as cysteic acid, which provides a highly reliable analysis for methionine that is converted to the sulfone.
Abstract: Publisher Summary A convenient determination of the combined content of cystine and cysteine in proteins depends on the oxidation of the amino acids to cysteic acid. This chapter discusses the determination of cystine as cysteic acid. A variety of strong oxidizing agents accomplish this reaction. The most suitable reagents are hydrogen peroxide and various peracids, particularly performic acid. The protein is subjected to acid hydrolysis and cysteic acid is determined by chromatography. This method provides a highly reliable analysis for methionine that is converted to the sulfone. The methods used are performic acid reagent, oxidation, hydrolysis, chromatography, methionine sulfone, and calculations. The quantity of protein required for a single determination will depend on the method used for the chromatography of cysteic acid. The cystine content may be expressed on a weight basis. It is convenient to express the cysteic acid and methionine sulfone values relative to a stable amino acid, such as alanine or leucine, determined in the same chromatogram.

823 citations


Book ChapterDOI
TL;DR: This chapter discusses the performic acid oxidation, a standard technique in protein chemistry with insulin for the cleavage of disulfide bonds in proteins, which will oxidize phenolic groups and the hydroxyl functions of serine and threonine side chains.
Abstract: Publisher Summary This chapter discusses the performic acid oxidation. The scission of disulfide bonds by oxidation with performic acid is a standard technique in protein chemistry with insulin. The virtue of the procedure is that cysteic acid and methionine sulfone, the oxidation products of cystine and methionine contains sulfur in a stable oxidation state. In addition to tryptophan and the sulfur-containing amino acids, it will oxidize phenolic groups and the hydroxyl functions of serine and threonine side chains. Conversion of cystine to cysteic acid usually proceeds in a yield of 85–90%, while oxidation of methionine to the sulfone is essentially quantitative. Since the reaction is usually conducted with a large excess of reagent, removal of this excess from the protein must be accomplished under mild conditions if over oxidation is to be minimized. Conditions suitable for the oxidation of a protein or peptide should be determined in each individual instance with the aid of amino acid analyses. Performic acid reagent, oxidation of ribonuclease, and removal of halide ion are also focused. A significant limitation to the use of performic acid oxidation for the cleavage of disulfide bonds in proteins is the concomitant destruction of tryptophan.

812 citations


Book ChapterDOI
TL;DR: The reaction is applied in a number of ways, such as in the structural elucidation of peptides and proteins, the detection of multiple forms of enzymes and of oxidized residues of methionine, the preparation of physiologically active peptide fragments, the location of newly introduced cross-linkages in enzymes, and the identification and characterization of fragments from enzyme active sites.
Abstract: Publisher Summary This chapter discusses the applications of the cyanogens bromide reaction Cyanogen bromide is capable of cleaving thioethers The action of cyanogen bromide upon proteins is unique in its selective attack on methionine The reaction of methionine with cyanogen bromide is greatly facilitated by the strong neighboring group effect exerted by the carboxyl group Cyanogen bromide is synthesized from bromine and potassium cyanide The selectivity of the reaction of cyanogen bromide with amino acids depends on pH The selectivity of the cyanogen bromide reaction is demonstrated by exposure to cyanogen bromide of a standard mixture of amino acids, as is used for calibration purposes in automated amino acid analyzer systems A number of applications to the general structural elucidation of peptides and proteins are: ribonuclease, rabbit γ-globulin, and chymotrypsin The reaction is applied in many other instances and is useful in a number of ways, such as in the structural elucidation of peptides and proteins, the detection of multiple forms of enzymes and of oxidized residues of methionine, the preparation of physiologically active peptide fragments, the location of newly introduced cross-linkages in enzymes,and the identification and characterization of fragments from enzyme active sites

743 citations


Book ChapterDOI
TL;DR: In this article, the authors discuss the formation and stability of dansyl chloride procedure and its use as a means of studying the α-amino and other reactive groups of proteins and peptides.
Abstract: Publisher Summary This chapter discusses the formation and stability of dansyl chloride procedure Dansyl chloride is used for providing fluorescent “handles” for the study of proteins The reaction of the dye with α-chymotrypsin led them to propose its use as a means of studying the α-amino and other reactive groups of proteins and peptides It is useful in determining the amino acid sequences of small amounts of peptides Dansyl chloride is a typical aromatic sulfonyl chloride, and reacts with a wide variety of bases, forming derivatives of differing stabilities Some recommended procedure for peptides discussed are (1) reagents, buffers, and marker mixtures, (2) labeling of peptide, (3) hydrolysis, (4) visualization of compounds on paper, and (5) interpretation of results The increased requirement for dansyl chloride brings with it the necessity for removing the large amounts of DNS-OH formed by its hydrolysis The removal procedures used are labeling, removal of salt, urea, DNS-OH, hydrolysis, and identification of end groups Some of the advantages and limitations of the method are mentioned The main advantage is the ease and rapidity with which end groups may be determined on very small amounts of peptides

597 citations


Book ChapterDOI
TL;DR: This chapter discusses the preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver, which is purified from microsomes both as a partially purifiedmicrosomal subparticle and with lipase treatment and fractionation, as a soluble flavoprotein essentially homogeneous in the ultracentrifuge.
Abstract: Publisher Summary This chapter discusses the preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver. It is purified from microsomes both as a partially purified microsomal subparticle, and with lipase treatment and fractionation, as a soluble flavoprotein essentially homogeneous in the ultracentrifuge. The assay depends upon measurement of the rate of cytochrome c reduction at 550 mμ. Cytochrome c reductase activity in microsomes is associated with marked TPNH-neotetrazolium diaphorase activity. This activity is disproportionately lost upon lipase treatmeat of microsomes, and its loss may serve as an indication of loss of an intermediate cofactor in the intact microsome, or of loss of a specific environmental or configurational state of the enzyme. The steps of purification procedure described are: preparation of lipase, preparation of microsomes, lipase solubilization, pH precipitation, ammonium sulfate fractionation, column chromatography on hydroxylapatite, and calcium phosphate gel concentration. The prosthetic group of TPNH-cytochrome c reductase enzyme is flavin adenine dinucleotide, but the apoenzyme can be reactivated by FMN and FAD. The enzyme is specific for TPNH, but is relatively nonspecific for electron acceptor. TPNH-cytochrome c reductase has a pH optimum of 8.2, which appears to be independent of the buffer used. TPNH-cytochrome c reductase is markedly stimulated by low levels of p-chloromercuribenzoate; maximal activation is reached at 2 moles of PCMB per mole of flavin, and higher concentrations inhibit.

556 citations


Book ChapterDOI
TL;DR: DT diaphorase is a flavin adenine dinucleotide (FAD)-eontaining flavoprotein, which catalyzes the oxidation of NADH and NADPH by various dyes and quinones with a maximal velocity of the order of 10 7 moles per mole of flavin per minute.
Abstract: Publisher Summary DT diaphorase is widely distributed in the animal kingdom, and is a flavin adenine dinucleotide (FAD)-eontaining flavoprotein, which catalyzes the oxidation of NADH and NADPH by various dyes and quinones with a maximal velocity of the order of 10 7 moles per mole of flavin per minute. Bovine serum albumin (0.07%), polyvinylpyrrolidone (5%), and certain nonionic detergents such as Tween-20 or -60 (0.2%) are activators of DT diaphorase; the concentrations in parentheses are those required for maximal activation. The activation is reversible and implies both an elevation of the V max of the enzyme and an increase of its affinity for NADH and NADPH. The need for an activator increases during the purification and storage of the enzyme, probably because of the removal or destruction of a naturally occurring activator. Various electron acceptors for DT diaphorase are listed. 2, 6-Diehlorophenolindophenol (DCPIP) and certain benzo- and naphthoquinones are the best electron acceptors, whereas methylene blue and ferricyanide are relatively inefficient, and cytochromes c and b 5 are practically inactive as electron acceptors. The chapter also describes the assay and purification procedure of DT diaphorase, and discusses the DT diaphorase relation to other diaphorases and related enzymes.

526 citations


Book ChapterDOI
TL;DR: This chapter discusses the preparation of succinated dehydrogenase and reconstitution of succinate oxidase and the properties that are for succinate dehydrogen enzyme from bovine heart.
Abstract: Publisher Summary This chapter discusses the preparation of succinate dehydrogenase and reconstitution of succinate oxidase Succinate dehydrogenase is solubilized by butanol from the Keilin-Hartree heart muscle preparation, preincubated with a moderate concentration of succinate The soluble dehydrogenase is reactive with the cytochrome system This method is also applicable to other starting material, such as heart and liver mitochondria, but less effectively Another method for preparing succinate dehydrogenase active to the cytochrome system is by means of the alkaline cleavage of the heart muscle preparation pretreated with succinate The activity of soluble succinate dehydrogenase is assayed by the technique of reconstitution In the presence of the cytochrome particle, the dehydrogenase is incorporated into the particle to form an active succinate oxidase Reconstituted succinate oxidase catalyzes the oxidation of succinate by oxygen to fumarate and water just as intact oxidase does Conventional methods, such as manometry, are then used for the determination Succinate dehydrogenase can also catalyze the oxidation of succinate by a number of artificial electron acceptors For isolation of reconstituted succinate oxidase, it is more convenient to use an excess amount of the dehydrogenase The chapter also discusses the properties that are for succinate dehydrogenase from bovine heart

516 citations


Book ChapterDOI
TL;DR: Thin-layer separation methods for nucleic acid derivatives that are found effective in laboratory are described, including PEI-cellulose anion-exchange layers, which afford particularly sharp and highly reproducible separations of nucleotides.
Abstract: Publisher Summary The chapter describes thin-layer separation methods for nucleic acid derivatives that are found effective in laboratory. Thin-layer methods are developed for the separation and quantitative determination of nucleic acid bases, nucleosides, and nucleotides. The main advantages of thin-layer methods are sharpness of resolution, great sensitivity, simplicity, and speed. Two-dimensional anion exchange thin-layer chromatography allows resolving complex mixtures of nucleotides that are difficult to separate on paper or on a single column. Layers of the binder-free cellulose powder MN 3002 are well suited for qualitative and quantitative work. Avicel-cellulose powder is also used. A multitude of cation- and anion-exchange celluloses for thin-layer chromatography is commercially available. PEI-cellulose anion-exchange layers afford particularly sharp and highly reproducible separations of nucleotides, if they are prepared in the laboratory from unmodified cellulose and poly (ethyleneimine). For quantitative assays of bases and nucleosides, compounds are transferred from the layer to a paper wick, eluted from the paper, and measured spectrophotometrically. In combination with the quantitative techniques described, chromatography on PEI-cellulose layers are used to assay incubation mixtures and biological extracts. Oligonucleotides are separated by thin-layer chromatography or thin-layer electrophoresis or by two-dimensional combination of these methods.

Book ChapterDOI
TL;DR: This chapter discusses the principle, method, and procedure of determination of the tryptophan content of proteins with NBS, utilizing the reagent N -bromosuccinimide (NBS).
Abstract: Publisher Summary A rapid and convenient spectrophotometric method for the estimation of tryptophan in proteins, utilizing the reagent N-bromosuccinimide (NBS) is discussed This chapter discusses the principle, method, and procedure of determination of the tryptophan content of proteins with NBS Their method of assay makes application of the fact that the indole chromophore of tryptophan, absorbing strongly at 280 mμ is converted on oxidation with NBS to oxindole, a much weaker chromophore at this wavelength The oxidation is conducted in an acidic medium, usually an acetate or acetate-formate buffer, with carefully controlled amounts of NBS Some proteins may possess tryptophan residues which prove refractory to NBS oxidation either reacting very slowly under the usual conditions This behavior probably reflects tryptophan residues buried in hydrophobic regions of the protein Useful information on the environment of the tryptophan residues in a protein can be obtained by comparison of titrations carried out in the presence and absence of urea

Book ChapterDOI
TL;DR: This chapter investigates the preparation, properties, and conditions for assay of mitochondria, and three procedures are described for the isolation of mitochondira from slaughterhouse material.
Abstract: Publisher Summary This chapter investigates the preparation, properties, and conditions for assay of mitochondria. Three procedures are described for the isolation of mitochondria from slaughterhouse material. These procedures for the isolation of beef heart mitochondria differ only in the manner in which the heart mince is homogenized. The fractionation of the mitochondria and washing steps are the same in the three procedures. Procedure 1 dislodges the loosely packed damaged mitochondria, and the mixture is decanted and discarded. A portion of the light beef heart mitochondria (LBHM) adheres to the wall of the centrifuge tube, and it can be removed with the aid of a glass-stirring rod. Procedure 2 involves the use of a proteolytic enzyme to aid in the homogenization of the heart mince, and has the advantage of producing a higher yield of mitochondria than the first procedure. Mitochondria prepared by this procedure manifest higher respiratory control ratios. Procedure 3 leads to the formation of a large proportion of damaged mitochondria (LBHM) but has the advantage that large amounts of material can be worked up at one time. This method uses a Waring blendor to homogenize the heart mince. Mitochondria that are prepared and assayed with distilled water that had been passed through two ion exchange beds consistently exhibit P : O ratios greater than 3 for pyruvate, β-hydroxybutarate, α-ketoglutarate and greater than 2 for succinate.

Book ChapterDOI
TL;DR: This chapter describes the procedure and applicability of the sequential degradation plus dansylation method, used extensively in several investigations of the amino acid sequences of proteins, notably that of α-chymotrypsin.
Abstract: Publisher Summary This chapter describes the procedure and applicability of the sequential degradation plus dansylation method. This procedure is used extensively in several investigations of the amino acid sequences of proteins, notably that of α-chymotrypsin. All normal protein amino acids are encountered, and with few exceptions provided no difficulties. The yields of dansyl end groups decline slowly during successive stages of the degradation, and it is necessary to progressively increase the size of sample used for reaction. The method employs direct identification of the end groups with the very sensitive dansyl chloride technique. Approximately 1-5 millimicromoles are required for each step of the degradation, so that 20 millimicromoles is usually sufficient to establish the complete sequence of a penta- or hexapeptide. The limiting factor is the capacity of the electrophoresis equipment used in identifying end groups.

Book ChapterDOI
TL;DR: The extent to which complete reduction and S-carboxymethylation have been attained is determined by amino acid analysis and serves as a check on the extent of side reactions such as sulfonium salt or sulfoxide formation from methionine and modification of histidine or lysine.
Abstract: Publisher Summary This chapter reviews the reduction and S-carboxymethylation of proteins. Of the reagents available for the reduction of disulfide bonds, one of the most satisfactory is β-mercaptoethanol. The reduction in the absence of oxygen is performed with a large excess of reagent at a slightly alkaline pH and under conditions in which the protein is unfolded. Concentrated urea solutions, 8 or 10 M, have proved suitable as solvents. The carboxymethylation reaction generates iodide ions. To prevent the light-catalyzed oxidation of iodide to iodine, it is essential to perform the carboxymethylation step and the subsequent removal of reagents such as salts in the dark. Reduced carboxymethylated proteins may be isolated by procedures similar to those used for the reduced proteins. Many reduced carboxymethylated proteins are insoluble in water and can be obtained from the reaction mixture by dialysis. The extent to which complete reduction and S-carboxymethylation have been attained is determined by amino acid analysis. This also serves as a check on the extent of side reactions such as sulfonium salt or sulfoxide formation from methionine and modification of histidine or lysine. The chapter also discusses the reduction and S-carboxymethylation of ribonuclease A. Howevere, S-carboxymethylcysteine is determined with an amino acid analyzer.

Book ChapterDOI
TL;DR: This chapter describes the preparation and properties of reduced coenzyme Q-cytochrome c reductase (complex III of the respiratory chain), which can be stored as a solution in the Tris-suerose-histidine buffer for weeks at –20° with little loss in activity.
Abstract: Publisher Summary This chapter describes the preparation and properties of reduced coenzyme Q-cytochrome c reductase (complex III of the respiratory chain) The rate of reduction of cytochrome c by reduced coenzyme Q 2 is estimated from the amount of cytochrome c that is reduced (absorbency change at 550 mμ) in a sample of the assay mixture that has been allowed to react for 10 seconds The enzyme-catalyzed reaction is stopped by the addition of an appropriate stop reagent Three procedures for the preparation of (reduced coenzyme Q) cytochrome c reductase are described All three procedures utilize the S-1 fraction of the procedure described for the preparation of DPNH cytochrome c reductase The purification procedure described in the chapter utilizes the final supernatant solution obtained in the preparation of DPNH-cytochrome c reductase (Reduced eoenzyme Q)-eytoehrome e reduetase can be stored as a solution in the Tris-suerose-histidine buffer for weeks at –20° with little loss in activity The reduced forms of coenzyme Q 1 , eoenzyme Q 2 , and eoenzyme Q 10 all serve as electron-donor substrates with complex III (Reduced coenzyme Q)-cytochrome c reductase is inhibited almost completely by antimycin A at concentrations approaching the concentration of cytochrome c l in the solution of enzyme (15 μg per milligram of protein of complex III) In the assay system described, (reduced coenzyme Q)-cytochrome c reductase is active in the pH range from 60 to 85, with an optimal activity at pH 74

Book ChapterDOI
TL;DR: This chapter describes the preparations and properties of soluble NADH dehydrogenases from cardiac muscle, derived from the same segment of the respiratory chain, and the properties and methods of purification of these enzymes are discussed.
Abstract: Publisher Summary This chapter describes the preparations and properties of soluble NADH dehydrogenases from cardiac muscle. The known soluble NADH dehydrogenases can react only with artificial electron acceptors, cytochrome c, and lower homologs of coenzyme Q. The behavior of cytochrome c in the reaction with the soluble preparations is probably different, from that with NADH dehydrogenase in the particulate form, for example, ETP or the Keilin-Hartree preparation. Among the artificial acceptors, 2, 6-dichlorophenolindophenol (DCIP), and ferricyanide are widely used. These reactions are conveniently followed spectrophotometrically. The activities are either expressed as NADH oxidized at a fixed concentration of the acceptor or extrapolated to its infinite concentration (V max ). The methods for the preparation of the 37° enzyme, the 30° enzyme, the enzyme reported by Mackler et al., and the thiourea enzymes are described. The properties and methods of purification of these enzymes are also discussed. Although these soluble NADH dehydrogenases differ in varying degrees in many aspects, these are derived from the same segment of the respiratory chain. The nature of the soluble enzyme isolated is dependent on the method and conditions of solubilization employed.

Book ChapterDOI
TL;DR: This chapter examines the titration behavior of proteins and the preferred denaturing agent is guanidine hydrochloride, because of its greater ability to unfold proteins than other reagents, such as urea, and its stability in solution.
Abstract: Publisher Summary This chapter examines the titration behavior. The titration behavior of a protein is similar to that of a mixture of the constituent amino acids with their α-amino and α-carboxyl groups blocked, except for the terminal amino acids. The information obtained from titration curves of enzymes is based on an analytical nature. However, there is an important difference between the titration of a protein and the corresponding mixture of blocked amino acids. An electrometric titration curve represents the relation between pH and the number of moles of protons bound by, or removed from, 1 mole of protein in the reaction. The isoionic point depends on the protein concentration. Spectrophotometric titration of tyrosine groups, thermodynamic interpretation, and group counting is also discussed. The natural choice is the titration curve in a denaturing medium in which the protein is in the unfolded form, with virtually all titratable groups exposed to the solvent. The preferred denaturing agent is guanidine hydrochloride, because of its greater ability to unfold proteins than other reagents, such as urea, and its stability in solution. The result obtained from the titration curve of native proteins is also provided.

Book ChapterDOI
TL;DR: This chapter discusses the application of inhibitors and uncouplers that are useful in the study of oxidative phosphorylation, and the most commonly used uncoupler is 2, 4-dinitrophenol, which affects all phosphorylating sites in the respiratory chain, but has no effect on the substrate-linked phosphorylations.
Abstract: Publisher Summary This chapter discusses the application of inhibitors and uncouplers that are useful in the study of oxidative phosphorylation. The various inhibitors and uncouplers may be classified based on the chemical-coupling theory of oxidative phosphorylation, which can be formulated. The chapter discusses the non-site-specific inhibitors (oligomycin and aurovertin), site I-specific inhibitors (Amytal and alkylguanidines), site II-specific inhibitors (n-Heptylquirwline N-oxide and phenylethylbiguanide), site III inhibitors (synthalin), and inhibitor of phosphorylafion of added ADP. Oligomycin is used to inhibit the synthesis of ATP by respiratory-chain oxidative phosphorylation without inhibiting the initial conservation of energy. Aurovertin is used in ethanolie solution, and 0.4 micromole aurovertin per gram protein inhibits oxidative phosphorylation at all sites. As inhibition by Amytal is partially relieved by dinitrophenol, this compound may be classed as an inhibitor of oxidative phosphorylation. The most commonly used uncoupler is 2, 4-dinitrophenol, which affects all phosphorylation sites in the respiratory chain, but has no effect on the substrate-linked phosphorylation. 2,6 -Dinitrophenol acts very similarly to 2, 4-dinitrophenol. The concentrations of 2, 6-dinitrophenol, at different pH's, inducing the maximal ATPase and maximal stimulation of respiration in the absence of ADP and phosphate. Dicoumarol [3, 3 ' -methylenebis (4-hydroxycoumarin)] is active on all phosphorylating sites of the respiratory chain, and has no effect on the substrate-linked phosphorylation. Arsenate readily uncouples substrate-linked phosphorylations but is relatively ineffective against respiratory-chain phosphorylation. Other uncouplers gramicidin and valinomycin are also discussed.

Book ChapterDOI
TL;DR: In this paper, the authors focus on some of the possibilities and future potentials inherent in fluorescence methods as well as the difficulties in making absolute measurements and obtaining corrected spectra.
Abstract: Publisher Summary This chapter focuses on some of the possibilities and future potentials inherent in fluorescence methods as well as to some of the difficulties in making absolute measurements and obtaining corrected spectra. Fluorescence involves the absorption of energy by a molecule with a transition from the ground state to one of several excited states followed by rapid internal conversion to the lowest energy excited state and finally by a return to the ground state with the emission of light. Fluorescence measurements are usually more selective than spectrophotometry. The chapter also describes the instrumental aspects of fluorescence, a typical fluorometer, and detailed information about the components utilized in building such an instrument. The specific components are light sources, igniters, light-source housings, monochromators, and detectors. A number of commercial fluorometers and spectrophotofluorometers are also discussed. The potential procedure for obtaining corrected spectra involves the use of digital computing facilities. Although some of the instruments give partially corrected spectra, it is important to understand the errors involved in fluorescence measurements and the type of corrections that must be applied in most cases.

Book ChapterDOI
TL;DR: A combined procedure results in morphologically well defined outer and inner membrane fractions with a quantitative recovery that makes it suitable for the study of intramitochondrial distribution of enzymes and other chemical constituents.
Abstract: Publisher Summary This chapter describes the separation and enzymatic properties of the inner and outer membranes of rat liver mitochondria. A brief exposure of isolated rat liver mitochondria to sonic oscillation, followed by centrifugation on a sucrose gradient, resulted in the separation of a particulate light subfraction from the bulk of the mitochondria, which exhibited a high rotenone-insensitive NADH-cytochrome c reductase activity, but was devoid of rotenone-sensitive NADH-cytochrome c reductase and other respiratory chain-linked enzyme activities. A similar subfraction was obtained when the mitochondria were subjected to swelling and contraction—rather than sonication—prior to density gradient centrifugation. As both the sonication and the swelling-contraction procedure yielded only partial separation of the rotenone-insensitive NADH-cytochrome c reductase, a combined procedure is described. This results in morphologically well defined outer and inner membrane fractions with a quantitative recovery that makes it suitable for the study of intramitochondrial distribution of enzymes and other chemical constituents. Electron microscopic examination of the heavy and light submitochondrial fractions reveals the following: Osmium-fixed specimens of the heavy subfraction consist of relatively large vesicles bordered by a single membrane. Negatively stained specimens of the same subfraction show mitochondrial images in the stage of bursting, with protrusions of unfolding cristae. The osmium-fixed light subfraction consists of relatively small vesicles (average diameter 0.2 μ ) bordered by a single membrane and devoid of inner structures.

Book ChapterDOI
TL;DR: This chapter describes the preparations of succinate-cytochrome c reductase and the cytochrome b-c 1 particle, and reconstitution of succenic dehydrogenase, and spectrophotometrieally determined the activity of cy tochrome c or DCIP following the reduction.
Abstract: Publisher Summary This chapter describes the preparations of succinate-cytochrome c reductase and the cytochrome b-c 1 particle, and reconstitution of succinate-cytochrome c reductase. The principle of the preparations is based on the sequential fragmentation of the respiratory chain. The immediate aim is not to purify the fragmented particle, but rather to cleave the chain systematically into segments as structurally complete as possible. For the preparation succinate-cytochrome c reductase, the heart muscle preparation is made from sand-grinding and differential centrifugation. This preparation is suspended in phosphate–borate buffer, 0.1 M with respect to both components, and a protein concentration is adjusted to between 16 and 24 mg per milliliter. Succinate–cytochrome c reductase does not show cytochrome oxidase activity or catalyze the oxidation of DPNH by eytochrome c, DCIP, or ferricyanide. For preparation of the cytochrome b-c 1 particle, the succinate-cytochrome c reductase solution at about 10 mg protein per milliliter is adjusted to pH 9.5 at 0-4° with 1 N NaOH. Soluble succinate dehydrogenase or the eytochrome b-c 1 particle alone does not catalyze the oxidation of succinate by either cytochrome c or DCIP. The cytochrome b-c 1 particle in the presence of succinate dehydrogenase rapidly mediates the transport of hydrogen (electron) from succinate to cytochrome c or DCIP. The activity is then spectrophotometrieally determined by following the reduction of cytochrome c or DCIP. The determination of the cytochrome b-c l particle activity by reconstitution uses excess succinate dehydrogenase. For the isolation of reconstituted succinate-cytochrome c reductase, the scale may be extended and the concentration increased. The reconstitution reaction takes place within the time of manipulation.

Book ChapterDOI
TL;DR: This chapter describes the removal and binding of polar lipids in mitochondria and other membrane systems and the assay procedures for measuring the effect of lipid by lipid-requiring enzyme preparations are described.
Abstract: Publisher Summary This chapter describes the removal and binding of polar lipids in mitochondria and other membrane systems. The methodology described is divided into four categories: (1) methods for removal of lipid; (2) methods for preparing microdispersions of polar lipids; (3) assay of enzymatic activity in the presence of added phospholipid; and (4) methodology for rebinding phospholipid. The conditions for optimal stability and other properties of the enzymes have to be taken into account in the methodology for removal and reinsertion of the lipid. To evaluate whether an enzyme requires lipid for function, the preparation is treated to reduce its lipid content. The preparation, which is deficient in lipid, thus serves as the test system to which phospholipid can be added and reactivation of enzyme activity is measured. Sufficiently mild conditions have to be used for removal of the lipid to allow restoration of enzymatic activity with the readdition and rebinding of the lipid. Test systems for demonstrating a lipid requirement in a number of enzymatic activities of beef heart mitochondria are summarized. The assay procedures for measuring the effect of lipid by lipid-requiring enzyme preparations are described. The microdispersions of phospholipids allow a hydrophobic milieu to exist in an aqueous environment. This serves to explain in part the "solubilization" of neutral lipids in water in the presence of phospholipids.

Book ChapterDOI
TL;DR: The yield and increase of activity during the purification of the succinic-cytochrome c reductase is illustrated and the method of preparation as described in the chapter, beef heart mitochondria are isolated in 0.25 M sucrose and are frozen at –40° for a period of 1-7 days.
Abstract: Publisher Summary This chapter describes the preparation and properties of succinie–cytochrome c reductase (complex II - III). Assays of enzymatic activity are carried out at 38° in a Beckman Model DU spectrophotometer, equipped with a photomultiplier. The final reaction mixture contains ten micromoles phosphate buffer (0.10 ml); one micromole NaN3 (0.01 ml); 0.2 micromole EDTA (0.02 ml); five mg BSA (0.05 ml); 10 micromoles potassium succinate, pH 7.0 (0.10 ml); and water to a volume of 0.9 ml. The enzyme preparation to be assayed is diluted to a concentration of 100-200 μg protein per milliliter in a solution of 0.88 M sucrose 0.005 M in succinatc. The enzyme-catalyzed reduction of cytochrome c by succinate proceeds at a linear rate under standard conditions over the first minute. Then specific activity is calculated. The resulting increase in absorbancy at 550 mμ is followed during this linear phase. In the method of preparation as described in the chapter, beef heart mitochondria are isolated in 0.25 M sucrose and are frozen at –40° for a period of 1-7 days. The yield and increase of activity during the purification of the succinic-cytochrome c reductase is also illustrated.

Book ChapterDOI
TL;DR: The method is described that separates phosphomolybdate from ATP by extraction into butyl acetate and the general principle of purification procedure is to obtain a microsomal fraction by differential centrifugation of a sucrose homogenate and to form dispersed and activated particles with a detergent, urea, or a concentrated iodide solution.
Abstract: Publisher Summary The ATPase activity which requires Na+, K+, and Mg++ together and is inhibited by cardiac glycosides is a part of the enzyme system for the stoichiometric transport of Na+ outward and K+ inward across cell membranes. It is widely distributed in animal tissues and species. Organs, which transport Na+ and K+ to energize electrical activity or secretion, show much activity. The chapter examines the preparation, purification, and properties of sodium and potassium-stimulated ATPase. From the rate of release of inorganic phosphate from ATP in the presence of Na+, K++, and Mg++ is subtracted the rate of release in the presence of Mg++ and a cardiac glycoside inhibitor, such as ouabain. Released inorganic phosphate may be measured simply by adding 2.5 ml of 0.48 M HC104, mixing, filtering, and taking a 2.0-ml aliquot for assay. The method is described that separates phosphomolybdate from ATP by extraction into butyl acetate. The general principle of purification procedure described in the chapter is to obtain a microsomal fraction by differential centrifugation of a sucrose homogenate and to form dispersed and activated particles with a detergent, urea, or a concentrated iodide solution. Aging, freezing and thawing, and mild detergents may improve the activity or sensitivity of fresh homogenates or microsomal fractions. The Km for ATP is about 0.3 mM with Mg++ in slight excess. As the amount of Mg++ is reduced with maximal ATP, the activity decreases but the sensitivity increases. The Km for Na+ is about 1.5 mM and for K+ about 0.4 mM. The apparent affinity of the Na+-site for K+ is about 7-fold less than for Na+ whereas the apparent affinity of the K+-site for Na+ is about 160-fold less than for K+.

Book ChapterDOI
TL;DR: This chapter describes the preparation procedure, purification, and properties of the cytochrome c from vertebrate and invertebrate sources, and describes the methods used to extract the protein from ground and homogenized tissue.
Abstract: Publisher Summary This chapter describes the preparation procedure, purification, and properties of the cytochrome c from vertebrate and invertebrate sources. Cytochrome c is extracted from ground and homogenized tissue with a dilute solution of aluminum sulfate at pH 4.5. At slightly alkaline pH, excess aluminum ions are precipitated as the hydroxide, and exchanged for three monovalent ammonium ions. This permits the direct collection of the protein from the extract on a cation exchange resin. The cytochrome c is purified by (NH 4 ) 2 SO 4 fractionation, cation exchange chromatography, and crystallization. Other methods, involving extraction with trichloroacetic or sulfuric acids, extraction at neutral pH following the decomposition of the tissue in acetic acid or by treatment with a suerose-saponin solution and acetone are described. Cytochrome c is a basic protein with a molecular weight near 12,300. It consists of single polypeptide chain 103-108 amino acid residues long, to which a single heme prosthetic group is covalently attached by thioether bonds formed by the addition of the sulfhydryl groups of two cysteinyl residues in positions 14 and 17 across the vinyl side chains of the porphyrin ring. In the vertebrate cytochromes c the amino-terminal residue is acetylglyeine, while the proteins from nonvertebrate sources carry several extra residues in place of the acetyl.

Book ChapterDOI
TL;DR: This chapter describes various chemical methods used in the detection of peptides and the reaction of ninhydrin with peptides, which is a complex reaction at α- and ɛ-amino groups and hydrolysis.
Abstract: Publisher Summary This chapter describes various chemical methods used in the detection of peptides The reactions with ninhydrin and with the Folin-Lowry reagent are both used for the detection and estimation of peptides in solution The reaction of ninhydrin with peptides is complex because, in addition to reaction at α- and ɛ-amino groups, hydrolysis may also occur The rates of these processes are a function of the structure of the peptide The variability of color yield is decreased and the overall sensitivity of the procedure is increased when the peptide is first subjected to hydrolysis Three major chemical methods discussed are (1) ninhydrin reagent, (2) alkaline hydrolysis, and (3) ninhydrin reaction Ninhydrin reagent involves three preparation steps, namely––the methyl cellosolve, buffer solution 4M, pH 5 , and preparation and storage of reagent In alkaline hydrolysis, the reaction is performed in polypropylene test tubes and the samples to be analyzed are carefully pipetted into the bottoms of the tubes The procedure of the ninhydrin reaction is also discussed

Book ChapterDOI
TL;DR: This chapter discusses the procedure of peptide mapping by using two major techniques, namely––paper chromatography and electrophoresis, to afford a rough identification of differences in amino acid sequence between two closely related molecules.
Abstract: Publisher Summary The development of procedures for peptide mapping requires a technique that is capable of giving highly valuable information on the primary structure of protein molecules. This chapter discusses the procedure of peptide mapping by using two major techniques, namely––paper chromatography and electrophoresis. An enzymatic digest of a protein to chromatography is subjected, followed by high-voltage electrophoresis in the second dimension. The relative mobility of each peptide in the two dimensions depends on its composition and to a lesser extent its sequence. The procedure is designed to afford a rough identification of differences in amino acid sequence between two closely related molecules, such as residue replacements due to genetic mutation. Several solvent systems are used for peptide mapping and vary in applicability to different proteolytic digests. A variety of amino acid-specific reagents used to stain a peptide map and enhance its usefulness are focused. Many specific staining techniques found in standard texts dealing with chromatography and are helpful in technique of peptide mapping such as Ehrlich stain for indoles are discussed. Interpretation of the map, peptide mapping for preparative purposes, peptide mapping to evaluate digestion by exopeptidases, and technique of diagonal peptide mapping are also discussed in the chapter.

Book ChapterDOI
TL;DR: Formation of the intermediate is poorly reversible by ADP so that greater efficiency of incorporation of radioactivity could probably be obtained if the amount of carrier ATP were reduced.
Abstract: Publisher Summary This chapter discusses 32 P-labeling of a (Na + + K + )-ATPase intermediate, and describes the preparation of [γ- 32P ] ATP and phosphorylated intermediate. For the preparation of [γ- 32P ] ATP, the reaction is carried out at about 23° in the lead-glass bottle in which the 32 P comes. The reagents are added in the sequence given. Neutralization at step 2 is tested by adding a tiny drop to pH paper. Any pH between four and eight is satisfactory at this point. The cofactor solution (3) may be stored frozen. During the addition of the MgC1 2 (step 8), the reaction mixture is swirled gently in order to minimize the possibility of precipitating [ 32 P] MgNH 4 PO 4 . Formation of this precipitate delays labeling of ATP for about 10 hours. The enzyme preparations may provide enough P i and NH 4 + to form the precipitate if the procedure given is modified. For the preparation of phosphorylated intermediate, the reaction is carried out at 0° with rapid stirring. The reagents are added in the sequence given. Five seconds after the addition of the radioactive ATP, the reaction is stopped by the addition of 35 ml of 0.3 M HCI04 or trichloroacetic acid (fresh) containing 0.6 mM unlabeled ATP. The acid is at 0°, and the ATP is added to it less than 1 hour before the reaction is carried out. Formation of the intermediate is poorly reversible by ADP so that greater efficiency of incorporation of radioactivity could probably be obtained if the amount of carrier ATP were reduced.

Book ChapterDOI
TL;DR: This chapter discusses the microsomal lipid peroxidation that may be demonstrated by measuring (1) O 2 consumption, (2) NADPH disappearance, and (3) malonaldehyde (MA) formation.
Abstract: Publisher Summary This chapter discusses the microsomal lipid peroxidation that may be demonstrated by measuring (1) O 2 consumption, (2) NADPH disappearance, and (3) malonaldehyde (MA) formation. Results of an experiment involving the measurement of all three parameters are illustrated. In the experimental procedure, rat liver microsomes are prepared s by sedimenting the 10,000 g supernatant of a 0.25M sucrose homogenate of rat liver at 105,000 g for 60 minutes. O 2 consumption is measured with a Clark O 2 electrode. NADPH disappearance is monitored fluorometrically at 450 mμ with an excitation wavelength of 365mμ. An Eppendorf photometer with fluorometer attachment is a suitable instrument for this purpose. MA formed is measured colorimetrically with the thiobarbituric acid (TBA) reaction. O 2 consumption may alternatively be measured manometrically in the Warburg apparatus. Measurement of NADPH disappearance spectrophotometrically at 340 mμ is complicated by the turbidity of the microsomal suspension which, in addition, may change during the incubation. The maximal rate of NADPH-linked lipid peroxidation at 30° is approximately 160 millimicromoles of O 2 consumed per minute per milligram of microsomal protein. The NADPH disappearance accompanying the microsomal lipid peroxidation ranges between one-third and one-fifth mole of NADPH per mole of O 2 consumed. The maximal extent of lipid peroxidation is approximately one micromole of O 2 consumed per milligram of microsomal protein.