Showing papers in "Methods in Enzymology in 1981"

[44] Glutathione peroxidase

Abstract: Publisher Summary This chapter presents a procedure for the preparation of glutathione peroxidase, which is regarded as a major protective system against endogenously and exogenously induced lipid peroxidation. Two types of methods are used for determining the activity of glutathione peroxidase. One involves a direct measurement of unconsumed glutathione (GSH) at fixed time periods by polarographic GSH analysis' (Method 1), or by the dithionitrobenzoic acid method (Method 2). The second approach takes advantage of the capability of glutathione reductase, with nicotinamide adenine dinucleotide phosphate (NADPH), to regenerate GSH from oxidized GSH. The decrease in NADPH is continuously measured spectrophotometrically, while the GSH concentration in the enzymatic cycle remains essentially constant (Method 3). A convenient source for the preparation of glutathione peroxidase is bovine blood including the following steps: hemolysate; organic solvent precipitation; phosphate precipitation; absorption to phenyl-sepharose; and washing on diethylaminoethyl (DEAE)–sephadex, S-300 sephacryl, and hydroxylapatite column.

Assays for differentiation of glutathione S-transferases.

Abstract: Publisher Summary This chapter provides the spectrophotometric, titrimetric, nitrite, and cyanide assay for the differentiation of glutathione S-transferases. Spectrophotometric assays depend upon a direct change in the absorbance of the substrate when it is conjugated with glutathione (GSH). Because each of the reactions is catalyzed at a finite rate in the absence of enzyme, care is needed to reduce nonenzymatic catalysis by minimizing substrate concentrations and by decreasing pH wherever necessary. Titrimetric assay is based on the principle that the conjugation of alkyl halides with GSH can be measured titrimetrically. Although acid production accompanies many of the transferase catalyzed reactions in which thioethers are formed, titrimetry is only used when more convenient assays are not available. Nitrite assay is based on the principle that nitrite is released when GSH reacts with nitroalkanes or with organic nitrate esters. The nitrite can be assayed as the limiting factor in a diazotization reaction with sulfanilamide that produces a readily quantitatable pink dye. Cyanide assay is based on the fact that when glutathione transferases catalyze the attack of the glutathione thiolate ion on the electrophilic sulfur atom of several organic thiocyanates, it results in the formation of an asymmetric glutathionyl disulfide and cyanide. Cyanide can be readily quantitated by a calorimetric method.

Preparation of monoclonal antibodies: strategies and procedures.

Abstract: Publisher Summary This chapter discusses the strategies and procedures for the preparation of monoclonal antibodies (McAb). The derivation of permanent lines of hybrid cells, producing McAb, exhibiting certain desired properties, presents widely different degrees of difficulty. Desired properties include not only specific recognition of an antigen and other no less critical properties are the fine specificity of the antibody, avidity and kinetic parameters important for radioimmunoassays, cytotoxic properties necessary for direct complement-dependent lysis. The insoluble antigen and the antibody in the culture fluid are allowed to react. The free antibody is washed away. The amount of monoclonal antibody bound is measured directly or by binding of a second, labeled antibody capable of recognizing the first. Monoclonal antibodies can be easily labeled internally at high specific activity, using radioactive amino acid precursors. The choice of these is based on the efficiency of incorporation of labeled amino acids into secreted immunoglobulin in culture conditions. The hemagglutination assays are based on the ability of an antibody to agglutinate red cells, carrying the specific antigen. These assays have all the advantages in terms of extreme simplicity, speed, and direct visual reading of the results.

Cathepsin B, Cathepsin H, and cathepsin L.

Abstract: Publisher Summary This chapter describes two types of purification methods for Cathepsin B, Cathepsin H, and Cathepsin L. Method I is applicable to large amounts of frozen tissues, whereas method II is used with flesh tissue and takes advantage of a 50-fold purification factor attainable by isolation of lysosomes: it has the further advantage of separating the enzymes from inhibitors that are present in the cytosol and plasma. In first purification method, cathepsins B and H are purified from human liver. Method II involves purification of Cathepsins B, H, and L from rat liver. Method I include: extraction, autolysis, and acetone fractionation and DEAE-cellulose chromatography. The pool of cathepsin B from DEAE-cellulose is further purified by covalent chromatography on a column of aminophenylmercuric acetate coupled to Sepharose. Method II include: homogenization and cell fractionation gel; chromatography on Sephadex G-75; CM-Sephadex chromatography; chromatography of cathepsin L on concanavalin A-Sepharose. Cathepsin B can be with BZ-DL-Arg-NPhNO2 or Bz-Arg-2-NNap as substrate, wheras, Cathepsin H can be assayed selectively by use of an unblocked substrate such as Leu-NNap, Arg-NNap, or Arg-NMec. Three synthetic substrates have been used for cathepsin L assay: Bz-Arg-NH2, Z-Lys-OPhNO2, and Z-Phe-Arg-NMec.

Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples.

Abstract: Publisher Summary This chapter presents the assay of glutathione (GSH), glutathione disulfide (GSSG), and glutathione mixed disulfides (GSSR). The sum of the reduced and oxidized forms of glutathione can be determined by using a kinetic assay in which catalytic amounts of GSH or GSSG and glutathione reductase bring about the continuous reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) by nicotinamide adenine dinucleotide phosphate (NADPH). The sensitivity of the assay may be enhanced by measuring NADPH fluorometrically. There are other two enzymatic assay available. In the first assay, the formation of the reduced glutathione using glyoxalase I can be monitored directly at 240 nm, or at very low levels of GSH, a dual wavelength spectrophotometer can be used at the wavelength-pair 240–270 nm with the same extinction coefficient. The second assay involves O-phthaldialdehyde, which forms a fluorescent complex with GSH and can be monitored at 350 nm–420 nm.

Protein determination in membrane and lipoprotein samples: manual and automated procedures.

Abstract: Publisher Summary This chapter describes the protein determination in membrane and lipoprotein samples. A variety of methods have proved to be effective in estimating the protein content of water-soluble samples. The procedure used is based on this modified Lowry method. It is found that by adding sodium dodecyl sulfate to the alkali reagent, samples can be assayed directly without prior solubilization or delipidation. An increase in the copper tartrate concentration facilitates quantitation of protein in the presence of sucrose and EDTA. Color formation depends mainly on reduction of the Folin-Ciocalteu reagent by protein-bound copper and proceeds in two distinct steps. The wavelength used routinely in the modified Lowry procedure was chosen as a compromise between increased absorption of the final blue reduction product with longer wavelengths and the practical limitations of most spectrophotometers. The absorption peak of the blue chromophore extends through much of the visible spectrum in a broad plateau and reaches maximum at 750 nm. The effectiveness and rapidity of this modified Lowry procedure as compared to the original Lowry procedure for assaying complex biological systems are also discussed.

Glutathione transferase (human placenta).

Abstract: Publisher Summary Glutathione transferases with basic isoelectric points have been purified from the human liver, and an acidic form is purified from human erythrocytes. However, this chapter presents a procedure for the preparation of glutathione transferase from human placenta. Enzyme activity during purification is determined spectrophotometrically at 340 nm by measuring the formation of the conjugate of glutathione and l-chloro-2,4-dinitrobenzene. Purification procedure involve the extraction from the human placenta; the supernatant fraction from Step 1 is chromatographed on a column (12.5 x 80 cm) of Sephadex G-25 and the pooled fractions from Step 2 are applied to a column (9 x 13 cm) of DEAE-cellulose equilibrated with l0 mM Tris-HCl at pH 7.8. The pooled effluent from Step 3 is dialyzed overnight against 3 x 10 liter of 10 mM sodium phosphate buffer; then affinity chromatography is carried on S-hexylglutathione coupled to epoxy-activated sepharose 6B and the pooled fractions from Step 5 are charged onto a column of Sephadex G-75 that is equilibrated and eluted with 10 mM sodium phosphate at pH 6.7, containing 0.1 mM dithioerythritol. The active fractions of effluent are pooled.

Production of antisera with small doses of immunogen: multiple intradermal injections.

Abstract: Publisher Summary This chapter describes the production of antisera with small doses of immunogen. A wide variety of immunization techniques has been used to generate specific antisera in laboratory animals. Those techniques incorporate a variety of injection routes, vehicles, and frequencies of injection into appropriate laboratory animals. The immunogen is initially dissolved in a buffer at an appropriate pH and molarity to enhance solubilization of the immunogen in that aqueous solution. An equal volume of that solution is combined with Freund's complete or incomplete adjuvant. It is suggested that, when using Freund's complete adjuvant, which contains per milliliter 2 mg of heat-killed tubercle bacillus, one must be certain that the insoluble mycobacterium is suspended in the oil. One of the primary factors, governing the selection of the animal species, is the antigenic similarity between the substance to be injected and that present in the animal species to be injected. As a generalization, antibody titers are readily maintained or increased with booster injections, but the specificity of the antisera may be lost or markedly changed with repeated injections.

Glutathione S-transferases (rat and human).

Abstract: Publisher Summary This chapter presents a procedure for the preparation of glutathione transferases of the rat and the human. The glutathione S-transferases are the enzymes catalyzing conjugation reactions with glutathione as the first step in mercapturic acid synthesis. Although these enzymes may be distinguished from each other by their characteristic substrate-activity spectrum; all of the transferases are active with 1-chloro-2,4-dinitrobenzene as substrate. The reaction of this substrate and glutathione occurs spontaneously and the assay, therefore, requires the use of a control from which enzyme is absent. Not all of the data presented in the chapter summarizing the purification procedure were obtained with 1-chloro-2,4-dinitrobenzene. Methyl iodide, 1,2-dichloro-4-nitrobenzene, and 1,2-epoxy-3-(p-nitrophenoxy)propane have also been used. The homogeneous transferases prepared from human and rat liver represent a group that, aside from overlapping substrate specificity, resemble each other in size and subunit number. Although transferases A and C are similar to a greater degree, by reason of cross-reactivity to antibody raised against either protein, the other transferases from rat liver seem to be quite separate species.

Gamma-glutamyl transpeptidase.

Abstract: Publisher Summary This chapter presents a procedure for the preparation of γ- glutamyl transpeptidase In the assay of γ- glutamyl transpeptidase the most widely used substrate is L-y-glutamyl-p-nitroanilide p -Nitroaniline, released during transpeptidation and hydrolysis, is readily determined from the increase in absorbance at 410 nm A variety of other procedures has been used to determine the activity of γ-glutamyl transpeptidase Other chromogenic substrates include L-γ- glutamylanilide and L-γ-glutamylnaphthylamides Assay procedures in which glutathione is used are complicated by its oxidation to glutathione disulfide; oxidation can be retarded by including ethylenediaminetetraacetic acid in the reaction mixtures Convenient spectrophotometric assays involving the use of S -substituted glutathione derivatives are also available Such derivatives include S -pyruvoylglutathione and S -acetophenoneglutathione For purification procedures, various methods of solubilization are employed including the treatment of the particulate enzyme with detergents, organic solvents, and proteinases The rat kidney enzyme can be purified following its solubilization with either proteinases (eg, papain and bromelain) or detergents (eg, Triton X-100 and Lubrol WX)

Assay of peroxisomal beta-oxidation of fatty acids.

Abstract: Publisher Summary This chapter describes the assay of peroxisomal β-oxidation of fatty acids. In the presence of palmitoyl-CoA, the reduction of NAD to NADH that occurs at the third step of the β-oxidation spiral is measured spectrophotometrically. Assay method may be applied to mitochondria by omitting Triton X-100 and including final concentrations of 0.25 M sucrose, and 1 mM carnitine in the assay and using freshly isolated organelles diluted with 0.25 M sucrose. Under these conditions peroxisomes show somewhat submaximal activity. The spectrophotometric assay is strictly linear with the amount of enzyme and the results are obtained immediately. The radioactivity assay is more sensitive. The two assay methods give similar results in measuring the distribution of peroxisomal β-oxidation during cell fractionation experiments measuring the effect of hypolipidemic drugs on the activity of peroxisomal B-oxidation. It is found that peroxisomal β-oxidation does not require carnitine, is insensitive to freezing, and is not inhibited by 1 mM KCN, when assayed in homogenates or subcellular fractions.

Extraction of tissue lipids with a solvent of low toxicity.

Abstract: Publisher Summary This chapter discusses the extraction of tissue lipids with a solvent of low toxicity. The extraction of lipids from tissues is invariably done with volatile organic solvents, most of which are sold with warnings against excessive inhalation. For each gram of tissue, add 18 ml of extraction solvent and homogenize thoroughly. The mixture should be a well-dispersed suspension. After 30–60 s of mixing, filter the mixture, preferably with a sintered-glass, Buchner funnel under pressure rather than with a vacuum. Materials containing above-average water content, such as plasma, should be extracted with a larger volume of extraction solvent to keep the water in solution. In the case of plasma and whole blood, it is best to add the material to the solvent in small portions with continuous vortexing, if a fine suspension of the nonlipid portion is to be produced. The washing step is a convenient way to reduce the volume of the lipid extract, a factor to consider in working on a large scale. It removes primarily the higher boiling component, and thereby facilitates subsequent evaporative removal of solvent. Removal of solvent from hexane-isopropanol extracts is readily done by vacuum evaporation, but a warmer bath than usual is recommended.

Assay of coagulation proteases using peptide chromogenic and fluorogenic substrates.

Abstract: Publisher Summary Amino acid chromogenic and fluorogenic substrates have been used for many years for assaying proteases. The sensitivity of the assay procedures that employ these substrates and the convenience of spectrophotometric or fluorometric measurements has led to their widespread use. Most of the early amino acid chromogenic and fluorogenic substrates are highly selective for the primary specificity-determining (P1) amino acid; thus substrates such as benzoylarginine-p-nitroanilide, for assaying trypsin-like proteases, as well as aromatic amino acidp-nitrophenyl esters for chymotrypsin-like proteases, have been extensively investigated and employed for routine proteolytic enzyme assay. The recognition that both the selectivity of many proteases and their catalytic efficiency depend on interactions with subsite amino acids in the peptide substrate coupled with the availability of amino acid sequences around the cleavage sites in several zymogens of the coagulation and fibrinolytic systems has led to the synthesis and commercial availability of a variety of peptide chromogenic and fluorogenic substrates with much greater selectivity than the single amino acid chromogenic and fluorogenic substrates.

DNA analysis in the diagnosis of hemoglobin disorders.

Abstract: Publisher Summary This chapter deals with DNA analysis in the diagnosis of hemoglobin disorders. The use of restriction enzymes has proved to be a very powerful tool both for dissecting DNA sequences and for analyzing the way in which they are organized into coding and noncoding regions on chromosomes. Because these enzymes recognize specific nucleotide sequences and cleave the DNA at these sites, the resulting fragments can be fractionated according to size on electrophoretic gels. With radioactive complementary DNA probes, it is possible to focus on specific genes, such as the globin genes. DNA restriction fragments are electrophoresed on 0.8% agarose gels in Tris-acetate buffer, using a horizontal gel apparatus. The chapter describes the preparation of globin-specific hybridization probes. Purifying a sequence complementary to one of the globin genes has posed problems in the past, but several groups have prepared chimeric plasmids in which a double-stranded DNA copy of human globin mRNA has been inserted. After the isolation of the recombinant plasmid, the specific double-stranded sequence can be enzymatically labeled in vitro by nick translation.

Use of protein A-bearing staphylococci for the immunoprecipitation and isolation of antigens from cells.

Abstract: Publisher Summary This chapter describes the use of protein A-bearing staphylococci for the immunoprecipitation and isolation of the antigens from cells. Major developments have been made toward the molecular characterization of a great many cellular and viral proteins. The alternative approach to the precipitation of immune complexes is based on the substitution of chemically fixed, protein A-bearing strains of Staphylococcus aureus bacteria for the secondary antibody. The use of staphylococci for antigen isolation has several advantages over the double antibody method. Specific binding of immune complexes to the staphylococci is extremely rapid, occurring within seconds, and is stable in a variety of solvent systems, whereas nonspecific binding of the other proteins is low. The sequence of the steps for antigen isolation, with the staphylococcal adsorbent, includes the interaction of radiolabeled, solubilized antigen with a molar excess of specific antibody, the binding of immune complexes to the staphylococci, separation from other cell components by centrifuging and washing, and elution from the adsorbent for analysis. It is found that a variety of antigens can be isolated sequentially from the same radiolabeled cell extract with the appropriate antisera and staphylococci.

Alpha 2-macroglobulin.

Abstract: Publisher Summary This chapter presents the procedure for purification and assaying of α2-macroglobulin (M), which occurs in the plasma exclusively in the S form, which is active in the binding of proteins. The following procedure was developed with the aim of purifying a2M without any conversion into the inactive F form. For purification blood containing acid citrate-dextrose anticoagulant is obtained from a blood bank. The plasma is separated by centrifugation and precipitate is run through LKB Ultrogel AcA 34 and AcA 22. The peak of α2M in fractions early in the elution pattern is detected by gel electrophoresis, and fractions showing little contamination are combined, concentrated by ultrafiltration over a Sartorius type 12133 membrane. Further, the partially purified α2M is passed through a series of small columns. Finally, the solution of α2M is twice dialyzed and stored at 4°C, sometimes after freeze-drying. Two distinct types of assays are required in work with a2M: determination of total α2M, and of active α2M selectively. Total α2M is best quantified immunologically. Active α2M can be estimated by saturating it with trypsin, inhibiting all free trypsin with soybean trypsin inhibitor, and quantifying the bound trypsin with benzoylarginine nitroanilide as substrate.

Production and assay of antibodies to acetylcholine receptors.

Abstract: Publisher Summary This chapter describes the production and assay of antibodies to acetylcholine receptors All the basic methods necessary to assay and purify acetylcholine receptors (AChRs) and AChR subunits for use as immunogens to produce antisera and monoclonal antibodies (mAbs) to AChRs and to induce experimental autoimmune myasthenia gravis are discussed in the chapter AChR is routinely identified and quantitated, by the binding of radioactively labeled krait or cobra venom toxins, which are competitive inhibitors of acetylcholine binding An easy approach to study the specificity of antibodies to intact AChR is to test their reaction, with 123 I-toxin-labeled AChR, from various species by radioimmunoassay The competitive binding to native AChR method uses mAbs to AChR that have been mapped, by their ability to react, with denatured subunits to help map the subunit specificity of antibodies, that react only, with native AChR, and to distinguish between mAbs to various antigenic determinants on the same subunit It is demonstrated that the antigenicity of the native AChR molecule is dominated, by a small region, on the α-subunit

[8] Solid state nuclear magnetic resonance of lipid bilayers

Abstract: Publisher Summary This chapter discusses solid state nuclear magnetic resonance of lipid bilayers. The shortcomings of conventional high-resolution spectroscopy when applied to lipid bilayers are presented. The resolution in multiple-pulse experiments has been limited to 1–2 ppm and these techniques have been useful only for studies of small molecules. A method for obtaining high-resolution spectra of solids relies on the spatial factor to attenuate and is referred to as dilute spin spectroscopy. It is found that since the oscillating components are adjusted to the same frequency, the spin systems are in resonance and mutual spin flips can occur and heat can flow between the reservoirs, and they equilibrate to a common spin temperature. It is convenient to add an echo pulse to the experiment at the dilute spin frequency, as is shown in the chapter. The purpose of this pulse is to delay data acquisition until the receiver and filters of the spectrometer recover from the radiofrequency pulse. It is suggested that the sidebands can be used to obtain information on chemical shift and electric field gradient tensors.

Chemical depletion of glutathione in vivo.

Abstract: Publisher Summary The method for lowering tissue glutathione (GSH) levels involves the administration of compounds that react enzymically with GSH to form conjugates. The inhibition of GSH synthesis can also result in the depletion of this tripeptide in the organs with a sufficient turnover rate. The conversion of GSH to its oxidized form, glutathione disulfide (GSSG), has also been used to deplete GSH, mainly in isolated cell preparations; the choice of the depleting agent depends on the type of system under study. Diethyl maleate (DEM) remains one of the most useful compounds for depleting hepatic GSH in vivo. Intraperitoneal administration of DEM reduces the hepatic GSH levels of rats to 6-20% of control values in 30 min and for a period of 2 to 4 hr. Phorone is another α,β-unsaturated carbonyl compound that may be useful as a GSH depleting agent. As in the case of DEM, the conjugation of phorone with GSH is catalyzed by glutathione transferases.

Preparation of derivatives of ferrous and ferric hemoglobin.

Abstract: Publisher Summary This chapter describes the preparation of derivatives of ferrous and ferric hemoglobin. The preparation of the different derivatives can often be done directly from the hemolysate, without any further purification. This is justified by the fact that hemoglobin is the major proteic component of the erythrocytic cytoplasm. In other animal species, especially in fish, the hemoglobin composition consists of several components in comparable amounts, and purification of the individual components is necessary. From a strictly methodological point of view, as a general rule, very laborious procedures should be avoided to keep the protein in the native form; this is particularly true in the case of mutant hemoglobins exhibiting a reduced stability. A hemoglobin solution can be stored aerobically in the cold for a few days without appreciable changes of its properties. It is advisable to keep the protein in a concentrated solution and at neutral or slightly alkaline pH. A more prolonged storage of hemoglobin can be obtained in its deoxygenated form. However, there are technical difficulties in keeping the solution completely oxygen free, and this is an absolute requirement because partially saturated hemoglobin is more susceptible to autoxidation than is the fully oxygenated protein. The method of choice for keeping hemoglobin, as well as many other proteins, is rapid freezing and storage in liquid nitrogen.

[1] Preparation of blood hemoglobins of vertebrates

Abstract: Publisher Summary This chapter deals with the preparation of blood hemoglobins of vertebrates. The hemoglobins of vertebrates are the most extensively studied of any proteins, largely because of their ease of preparation. The red cells are easily separated from the plasma by centrifugation. Hemolysis leads immediately to a solution that is usually more than 90% pure. Nevertheless, precautions need to be taken to obtain preparations of maximum value. Hemoglobins from different organisms vary greatly in their stability and resistance to denaturation, tendency to oxidize to methemoglobin, solubility, and chromatographic and electrophoretic properties. What is appropriate for mammalian hemoglobins is frequently inappropriate for those of lower vertebrates. The chapter explains erythrocyte preparation. Blood from small animals is usually best obtained by cardiac puncture. Large animals can be bled from superficial veins—for example, those of the ears of rabbits and elephants. The caudal vein of fish is an excellent location for bleeding; it is usually easy, with a little practice, to insert a needle just ventral to the lateral line directly into the vein. The extraction of hemoglobin from very small animals and embryos presents special problems. The chapter also describes the preparation of hemolysate.

Preparation and properties of apohemoglobin and reconstituted hemoglobins.

Abstract: Publisher Summary This chapter describes the procedures for the isolation of globin in the native state and for its reconstitution with metalloporphyrins. Globins are isolated from the corresponding hemoproteins by the removal of the heme moiety at acid pH, under controlled experimental conditions. The quality of the globin samples obtained can be checked by titration experiments with hemin (ferric protoheme) and by studying the properties of the reconstituted hemoproteins. There are two known methods for the preparation of globin from the holoprotein; both are based on the decreased affinity of the heme for globin at acid pH values. The first method employs acid–acetone at low temperature to split the heme group from the globin, which precipitates and is subsequently redissolved in water. A second method is based on heme extraction in methyl ethyl ketone at acid pH; in this case, the apoprotein remains dissolved in the aqueous layer. Further purification of the globin is accomplished similarly in both methods. The chapter presents the physicochemical properties of globin.

A convenient and rapid cytopathic effect inhibition assay for interferon.

Abstract: Publisher Summary To meet the need for a rapid bioassay, a convenient microtiter assay based on reduction of cytopathic effect (CPE) is presents. The assay is quantitative, requires only 16 hr, and can be adapted to many cell and virus combinations in common use. For simplicity, the number of technical manipulations is reduced to a minimum. Various parameters affecting both speed and sensitivity are evaluated. The assay is developed primarily as a tool to aid in the purification of human leukocyte and fibroblast interferons; therefore, conditions have been optimized for the assay of these interferons. As an example, the standard procedure for assay of human leukocyte interferon with bovine MDBK cells and vesicular stomatitis virus (VSV) as the challenge virus is presented. It should be noted, however, that this general procedure is applicable for the assay of human fibroblast interferon and that other cell lines (WISH, FS-7, L cells), challenge viruses (Sindbis), and even human immune interferon can be used. A procedure for testing other cells and challenge viruses for applicability in the assay is outlined.

[52] Mammalian collagenases

Abstract: Publisher Summary This chapter presents the procedure for purification and assaying of mammalian collagenases, which proteinases acting at neutral pH to cleave the native helix of the interstitial collagens. Collagenase can be purified from the culture media of a variety of tissues or cells from different species. The purification steps include: ammonium sulfate precipitation; gel filtration with ultrogel AcA 44; DEAE-cellulose chromatography; and zinc chelate affinity chromatography. At the present time, only assay systems employing collagen itself as a substrate are suitable for the unambiguous detection of specific collagenases. Type I collagen is normally used as the substrate for the assay of collagenase and is prepared in soluble form from skin and tendon. Soluble collagen can be reconstituted into insoluble fibrils by incubating at 25-37°C at neutral pH and this provides the basis for the most widely used collagenase assays. Radiolabeled collagen fibrils are incubated with the enzyme and collagen degradation followed by the assay of radioactivity solubilized during the assay period.

Flame ionization detection applied to thin-layer chromatography on coated quartz rods.

Abstract: Publisher Summary This chapter describes the flame ionization detection (FID) applied to thin-layer chromatography (TLC) on coated quartz rods. Iatroscan combines TLC with the FID, and the TLC/FID combination extends TLC sensitivity by two or more orders of magnitude into the range achieved only with gas chromatography. A Chromarod, after development and freeing of solvent, is passed through the hydrogen flame and the carbon ions produced are collected and amplified. In the actual apparatus the housing supports a moving frame, which in the TH-10 model that accepts any number of Chromarods up to 10. The short development time of the Chromarod-S is an encouragement to the sequential use of different solvent systems. By judicious choice of solvent systems, complex separations can often be effected that are not possible with one solvent system, and others can be much improved. A complex mixture of hydrocarbon in a heavy fuel oil could be resolved on a Chromarod-S into four main fractions, which includes asphaltines and polar compounds, resins, polynuclear aromatics and alkyl aromatics, and aliphatics.

Polarized absorption and linear dichroism spectroscopy of hemoglobin.

Abstract: Publisher Summary This chapter is concerned with polarized absorption and linear dichroism spectroscopy of hemoglobin. Polarized absorption and linear dichroism are techniques that are used to study the optical properties of oriented systems. Unlike solutions, where light polarized in any direction is absorbed equally because the molecules are randomly oriented, the absorption of plane-polarized light by oriented molecules is dependent on the polarization direction of the incident light beam. Anisotropic absorption occurs because molecules fixed in space exhibit maximum absorption when the electric vector of the light is parallel to well-defined directions in the molecule. A polarized absorption experiment in which the spectra are measured in each of three orthogonal directions on a sample of known molecular orientation—such as a crystal of known structure—yields both the complete spectrum and the molecular direction of the transition moment for each absorption band. If, on the other hand, the transition moment directions are already established, the polarized absorption experiment along three orthogonal axes yields the molecular orientation. In a linear dichroism experiment, the difference between the optical densities in two directions is measured directly.

[29] Human plasminogen

Abstract: Publisher Summary Human plasminogen is known to possess multiple isoelectric forms, which can be isolated by isoelectric-focusing techniques. Glu-plasminogen 1 possesses multiple forms of pI values of 6.2, 6.3, 6.4, and 6.6. Glu-plasminogen possesses two isoelectric forms with pI values of 6.4 and 6.6. On the other hand, Lys77-plasminogen 1 contains three major isoelectric forms of pI 6.7, 7.2, and 7.5; whereas LysrT-plasminogen 2 contains three major forms of pI values 7.5, 7.8, and 8.1. Assays of plasminogen first require its conversion to plasmin by common activators. The conversion of human plasminogen to human plasmin is accomplished as a result of cleavage of the Arg-Val­-sl peptide bond. The enzyme is then assayed based on its ability to hydrolyze a-N-tosyl-Larginine methyl ester (TAME), utilizing a recording pH-stat to titrate the amount of acid, a-N-tosyl-L-arginine, liberated with time. Assays that rely on the spectrophotometric determination of the plasmin-catalyzed release of p -nitroanilide from the substrate H-D-valyl-L-leucyl-L-lysine-pnitroanilide dihydrochloride and on the determination of the plasmin-catalyzed release of p-nitrophenolate from N-Cbz-L-lysine-p - nitrophenyl ester 3° have also been fruitfully employed. Other assay methods for plasmin are available, such as the casein, azocasein, and fibrin plate assay.

Production of interferon in human leukocytes from normal donors with the use of Sendai virus.

Abstract: Publisher Summary This chapter describes the method used for the production of human leukocyte interferon The production of interferon is highly sensitive to changes in the incubation conditions Better yields are obtained in round flasks, for example, than in ordinary flat-bottom bottles The aeration seems to play an important role Attempts to improve production by bubbling air or CO 2 into the medium during the incubation have not been successful The cells must be kept in constant agitation If the stirrers are intentionally stopped at different times after induction, the production of interferon declines rapidly, although the settling of the cells to the bottom takes several hours The removal of most globulins by (NH 4 ) 2 SO 4 -precipitation does not affect the activity of serum, and the reduction of antibodies to Sendai virus appears not only to lower the amount of the inducer virus needed but also to make the yields of interferon more consistent Factors such as priming, induction, temperature, pH, and incubation medium, affect the yield of interferon, but not the kinetics of the production in human leukocyte suspensions The pH range for optimum interferon production is between 72 and 76

[63] Inactivation of trypsin-like enzymes with peptides of arginine chloromethyl ketone

Abstract: Publisher Summary The value of affinity labels in the identification and characterization of proteases in their physiological environments has been clearly demonstrated by the use of tosyl phenylalanyl chloromethyl ketone, tosyllysine chloromethylketone, and alanyl peptide chloromethyl ketones. This chapter presents the use of arginine chloromethyl ketone in the development of selective-affinity labels for individual trypsin-like proteases that function in blood coagulation, fibrinolysis, and hormone processing. This group of enzymes functions by the hydrolysis of either one or two bonds of their physiological substrates, usually at arginyl residues. The chapter uses the approach of incorporating part of the sequence of the physiological substrate in the reagent, thus taking advantage of binding selectivity in both primary and secondary sites. The reactivity of the chloromethyl ketones with proteases is determined by incubating the protease with the affinity label, removing timed aliquots, and measuring residual enzymatic activity. The initial concentration ofprotease was selected so that the concentration of affinity label was at least 10 times greater than that of the enzyme.