Analysis Of Proteins In Forensics 861 Separation of Proteins by HPCE

Detailed discussion of the use of HPCE for separation of proteins are available in specialized reviews (Karger, 1989 Rohlicek and Deyl, 1989 Steuer et al., 1990 Mazzeo and Krull, 1991 Deyl et al., 1994a, 1994b Li, 1994). In general, three methods may be used for protein separation by HPCE. It is possible by exploiting the charge differences to separate proteins according to their effective charge in acid or alkaline media. Acid buffers are usually preferred for complex protein mixtures, even...

Histamine NMethyltransferase Fukuda et al 1991

Histamine V-methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to histamine to form N-methylhistamine. This assay is suitable for following activity during enzyme purification. Histamine and N-methylhistamine were separated on a weak cation exchange (TSK CM2SW) column (4 mm X 150 mm). The mobile phase was prepared by dissolving 5.25 g of citric acid and 10 g of imidazole in 880 mL of water, and then mixing the resulting solution with 200 mL of acetonitrile....

LJJ

Figure 7.5 (A) Separation of Hae III digested < X174 RF DNA, 50 ug mL in 20 mM NaCl. (B) Influence of presample injection of 0.1 M Tris-acetate, pH 8.3. First injection 10-second pressure injection of Tris-acetate second injection 20-second injection of digest. Reprinted with permission from van der Schans et al., Chromatogr A 680 511 (1994). is now no longer a linear function. To correct this, a double injection technique is employed, whereby an unknown and a standard are injected...

LaAminoadlpylLCysteinylDValine Synthetase White et al 1989

The synthetase that catalyzes the synthesis of the named peptide from l-a-aminoadipic acid, l-cysteine, and l-valine uses ATP as the energy source. The enzyme activity is found in the actinomycete Streptomyces clavuligerus. After the enzymatic reaction, the samples were oxidized with performic acid and then converted to the isoindole derivatives. The resulting sulfonate isoindole derivatives of was sepa rated on a Beckman Ultrasphere ODS column (4.6 mm x 45 mm, 5 im). The mobile phase was a...

AcetylCoAArylamlne MAcetyltransferase Manneus et al 1990 Thomas et al 1990

The N-acetylation of different aryl- and arylalkylamines, using acetyl-CoA as the acetyl donor, proceeds when catalyzed by acetyl-Co A arylamine N-acetyltransferase. A natural substrate is serotonin, which is converted to N-acetylserotonin in the pineal gland and other regions of the brain. Serotonin and V-acetylserotonin are separated on a Spherisorb ODS 2 column (3.2 mm X 150 mm). The mobile phase was a 3 1 mixture of 0.1 M sodium acetate (pH 4.75) and methanol. The column effluent was...

Hydrodynamlc Injection by Pressure Vacuum Application In

Almost all the automated instruments, injection is carried out by application of pressure for a fixed time onto the sample vial into which the injection end of the capillary is dipped, or by application of vacuum at the opposite end (Figs. 3.4, 3.5). Following Poiseuille's law, the amount of sample loaded will increase with an increase in pressure the sample concentration and the injection time and will decrease with increases in solution viscosity and the length of the capillary. 3.5.1.2...

Carbohydrate Metabolism 971 0Galactosldase Naol and Yagl 1981

3-Galactosidase ( 3-D-galactoside galactohydrolase, EC 3.2.1.23) catalyzes the release of galactose from a variety of substrates including glycosphingo-lipids. The galactosidase specific for the release of terminal galactose from glycosphingolipids was studied using a derivative of a galactocerebroside, l-O-galactosyl-2-N-DANS-sphingosine, as the substrate. The product of the 3-galactosidase reaction, TV-DANS-sphingosine, was measured by means of HPLC. The product was separated from the...

Power Supply

A high voltage power supply for CE should deliver voltages up to 30 kV and currents up to 200-250 iA. Because of the direction of the EOF, which in silica capillaries is usually toward the cathode, the common polarity is with the anode at the injection end of the capillary and the cathode close to the detection window. CE separations are generally carried out at constant potential, but constant current may be preferable, especially when the control of temperature of the capillary is not...

Forensic Analysis

Capillary electrophoresis with LIF detection has been applied in the analysis of genetic markers for human identification (McCord et al., 1993 Srinivasan et al., 1993a, 1993b). In the analysis of a locus containing short tandem repeats of 3 bp in the range of 250 or less, a morcointercalating dye, YO-PRO-1, was added to the buffer system of a polymer network. To improve separation efficiency, it was also necessary to add ethidium bromide to the run buffer. By means of voltage programming...

Heme Oxygenase Lincoln et al 1988

Heme oxygenase is a microsomal enzyme that catalyzes the first and rate-determining step in the degradation of heme. This assay measures bilirubin, which is produced by the consecutive actions of heme oxygenase and biliver-din reductase. This assay is based on the separation and quantitation of bilirubin. The column was a C18 Novapak radial compression column. Solvent A contained 49 0.1 M monobasic ammonium phosphate, 40 methanol, 10 acetonitrile, and 1 DMSO, adjusted with H3P04 to pH 3.6....

Preparation And Assay Of Activities In Intact Cells

In group I, we considered the question of obtaining an activity from a tissue or organ, from a biological fluid, or from cultured cells. The primary task in all these samples was the separation of the extracellular and cellular compartments. Next, the problem of separation of the different cell types within the cellular compartment was considered. In the section that follows, we will open the cell for a look inside. However, let us first consider briefly the surface of the intact cell and the...

Nucleic Acids And Their Constituents

Capillary electrophoretic separation of nucleic acids has been reviewed (Cohen et al 1987a Kuhr, 1990 Gebauer and Thormann, 1991). In nucleotide and nucleoside analysis, MEKC has been the method of choice, using SDS (Row et al. 1987 Cohen et al., 1987b Kasper et al., 1988), dodecyltrimethylammonium bromide, or hexadecyltriethylammonium bromide (Liu et al., 1989). Other applications concerning chemically modified nucleotides, nucleosides, and nucleobases can be found in papers by Lecoq et al....

Amlnopeptldase Mousa and Couri 1983

Aminopeptidases are involved in the metabolism of opioid peptides including enkephalins and 3-endorphins. An HPLC method was developed to measure the hydrolysis of these compounds by measuring the formation of tyrosylglycyl-glycine, tyrosylglycine, and tyrosine. The separation was accomplished by reversed phase HPLC (Ultrasphere ODS column) with a mobile phase of 50 miW sodium phosphate buffer (pH 2.1) with acetonitrile and methanol (90 5 5). The column was eluted isocrati-cally, and an...

WAcetyltransferase Martin and Downer 1989

N-Acetyltransferase uses acetyl-CoA to acetylate the amino moiety of arylal-kylamines. In mammalian pineal gland, this enzyme catalyzes the production of N-acetyl-5-hydroxytryptamine, which is the precursor of melatonin. It is also involved in the inactivation of monoaminergic neurotransmitters in insects. Substrates (p-octopamine, dopamine, or 5-hydroxytryptamine) were separated from their N-acetylated products on an Ultrasphere I.P. Qg column (4.6 mm x 250 mm, 5 m). The mobile phase was...

Overview

This chapter presents a strategy for the design of an HPLC assay system. Section 4.1, Setting up the Assay, focuses on the enzymatic reaction and the steps leading to the development of the assay. These steps are previewed in Table 4.1. Section 4.2, The Use of HPLC to Establish Optimal Conditions for the Enzymatic Reaction, discusses the procedure for monitoring the activity of an enzyme with the HPLC method and the use of HPLC assays to determine the parameters required for obtaining optimal...

Glutamlne Synthetase Glutamate Synthetase and Glutamate Dehydrogenase Martin et al 1982 Marques et al 1989

Depending on the organism, assimilation of ammonia takes place via the activities of glutamine synthetase, glutamate synthetase, and or glutamate dehydrogenase. The assays described, which measure glutamate and or glutamine formation and disappearance, were developed for use in situ and in vitro. In the HPLC assay method described by Martin et al. (1982), glutamic acid and glutamine are separated by reversed-phase HPLC after derivatization with o-phthaldialdehyde 2-mercaptoethanol (OPA). The...

Amino Acid Decarboxylase Kochhar et al 1989

The assay described for amino acid decarboxylase can be used to quantitate the substrates and products associated with the decarboxylation of arginine, aspartate, 2,6-diaminopimelate, histidine, glutamate, lysine, and ornithine. Kochhar et al. (1989) characterized an assay for glutamate decarboxylase activity. Glutamate and 4-aminobutyrate were separated on a Nucle-osil Cg column. The mobile phase was 13 mM trifluoroacetate and 1 mM 1-octanesulfonate. Detection was by postcolumn derivatization...

Dlpeptldase Horiuchl et al 1982

In a study of dipeptidase by Horiuchi et al. (1982), the tripeptide hippurylhisti-dylleucine (Hip-His-Leu) was used as a substrate, and the assay involved measuring the amount of hippuric acid released by the enzyme. The substrate was separated from the product by reversed-phase HPLC (Nucleosil 7 C 8) using a mobile phase of methanol-10 mM KH2P04 (1 1) adjusted to pH 3.0 with phosphoric acid. Detection was at 228 nm. The separation of the components of the reaction mixture is shown in Figure...

Application of HPLC to the Assay of Enzymatic Activities

This chapter describes the anatomy of an enzyme assay, focusing on the significance of separation and detection in the assay procedure. A classification of the methods used in the assay of enzymatic activities is developed, using the separation step as the criterion for the grouping. Having placed the high performance liquid chromatography (HPLC) method within this classification, we then examine the question of when to use it and discuss some strategies developed for its use. The chapter also...

With Franco Tagliaro Zdeneck Deyl and Ivan Miksfk

In this book, the focus is on the use of high performance liquid chromatography (HPLC) for the separation and detection of biochemical components produced from enzymatic reactions. But the purpose of the book is also to introduce HPLC and high performance capillary electrophoresis (HPCE) as methods applicable for separation and detection of a variety of components, even those not produced by enzymatic reactions. For example, HPLC and HPCE have shown great promise in forensics, the application...

Uridine Diphosphate Glucuronosyltransferase Matsul and Nagal 1980 To and Wells 1984

Uridine diphosphate glucuronosyltransferase catalyzes the transfer of glucuronic acid from uridine diphosphate glucuronic acid (UDPGA) to various Figure 9.67 Isocratic HPLC separation and UV absorption detection of UMP, UDP-Gal, and UDP as sequentially eluted. A mixture containing 500 pmol of each compound was injected. (From Hymes and Mullinax, 1984.) substrates, such as 4-nitrophenol (4-NP), Phenolphthalein (P), and testosterone. Matsui and Nagair (1980) separated the substrates and the...

Capillary Gel Electrophoresis CGE

Gel electrophoresis is the main separation technique used in biomedicine, biochemistry, and biotechnology. As SDS-polyarcylamide gel electrophoresis (SDS-PAGE), it is the standard method for the separation and characterization of proteins, DNA sequencing (polyacrylamide gels), and DNA fragment mapping (agarose gels). However, gel electrophoresis (in slab gels) has always remained a manual, time-consuming technique, and the results depend on the expertise of the operator. The separation...

Terminating the Reaction

In designing an assay for an enzyme, it is often necessary to introduce a termination step into the protocol (see Chapter 1). This is often done when protein is present in the incubation mixture at a concentration that would clog the column. There are a variety of ways to accomplish this termination process. Ideally, it would not be necessary to add reagents that might otherwise clog the column or alter its performance. For example, consider the changes that occur in the incubation mixture when...

Theoretical Plate Number and Resolution

Two parameters are used to describe performance in chromatography. These are theoretical plate number (N) and resolution ( ). The magnitude of N, an expression of efficiency, is a measure of the ability of the system to restrict analyte diffusion during the separation process. The larger the N, the better, and the more efficient, the system. N is calculated directly from information provided by position of a peak on the chromatogram by first dividing the migration time of this peak by the peak...

Tyrosine Hydroxylase Haavlk and Flatmark 1980 Naol et al 1988 Mandal et al 1992

Tyrosine hydroxylase is a monooxygenase that catalyzes the conversion of l-tyrosine to Dopa. The activity is found in peripheral and cholinergic neurons and chromaffin cells of the adrenal medulla. HPLC methods have been developed for the assay of this activity. In one method, the Dopa formed during the reaction was partially purified by ion-exchange and aluminum oxide chromatography and the amount present quantified by reversed-phase HPLC (ODS column). The mobile phase consisted of 0.1 M...

Monoamine Oxidase and Phenol Sulfotransferase Sim and Hsu 1990

Monoamine oxidase catalyzes the conversion of dopamine to 3,4-dihydroxy-phenylacetic acid and tyramine to 4-hydroxyphenylacetic acid. Phenol sulfo- transferase transfers sulfate from 3'-phosphoadenosine-5'-phosphosulfate to dopamine, 3,4-dihydroxyphenylacetic acid, and phenol. This assay is sufficiently sensitive to measure the activity of either enzyme in 5 mg of rat brain and liver. Radiolabeled products were separated from substrates by chromatography on a Merck Qg column (5 xm). The mobile...

Dipeptldyl Carboxypeptldase Angiotensin I Converting Enzyme EC 3415 Baranowskl et al 1982 Dolg and Smiley 1993

Hplc Collagenase

Angiotensin I converting enzyme is a dipeptidyl carboxypeptidase that cleaves angiotensin I to angiotensin II and the dipeptide histidyl-leucine (His-Leu). The enzyme is bound to the membrane of lung arterial endothelium and is involved in the renin-angiotensin system that regulates blood pressure and fluid balance. In the assay reported by Baranowski et al. (1982), the rate of formation of angiotensin II and the dipeptide His-Leu from the substrate angiotensin I was followed. The reactant was...

Typical Analytes Small Molecules

Microdialysis is a technique for recovering low molecular weight compounds. The size of the compound recovered is determined by the cutoff parameter of the membranes used for constructing the microdialysis probes. The polycarbonate membrane of 0.5 mm i.d. used for commercial probes manufactured by CMA Mikrodialysis has a cutoff of 20,000 Da. A 2 mm probe perfused at 2 nL min with Ringer's solution at ambient temperature will recover some 1 to 2 of a 5000 Da peptide from a quiescent medium....

Extracellular Space

Microinjection Pump Cma 200

The extracellular space comprises about 18 to 20 of the total tissue volume of most soft organs. This tissue compartment represents an important link between blood capillaries and cell soma for transport of molecules in both directions. The space is filled with the extracellular fluid ECF which, in turn, directly communicates with other body fluids such as lymph or cerebrospinal fluid CSF . The ECF is a glutinous buffered medium consisting of salts and low molecular weight compounds, as well as...

With Kathi J Ulfelder

The analysis of double-stranded DNA dsDNA fragments, such as those produced by the polymerase chain reaction PCR and enzymatic digestion, has led to considerable advances in molecular biology. Since its advent in 1985 Saiki et al., 1985 Mullis and Faloona, 1987 , PCR technology has been used to directly detect and quantify viruses, to track inheritance patterns in a family, to diagnose numerous genetic diseases, to identify individuals in forensic applications, and to aid in mapping the human...

Xx

Figure 8.1 MEKC of bulk heroin, heroin impurities, degradation products and adulterants. Conditions capillary, 25 cm X 50 mm i.d. voltage, 20 kV temperature, 40 C buffer, 85 m M SDS-8.5 m M phosphate-8.5 m M borate-15 acetonitrile, pH 8.5 detector wavelength, 210 nm. Peaks b, morphine c, 3-MAM d, 6-MAM e, acetylcodeine f, heroin g, ph nobarbital h, noscapine i, papaverine j, methaqua-lone. From Weinberger and Lurie, 1991, with permission. migration times based on suitable internal standards IS...