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...

Vertebrate Collagenase Gray and Sanell 1982

In both the ai and a2 chains of type I collagen, degradation of the protein begins with cleavage of the Gly-Ile or the Gly-Leu bond by collagenase. In the assay developed for this activity, the collagen was replaced by the peptide dinitrophenyl During the course of the reaction, this substrate was cleaved into the two products DNP-Pro-Gln-Gly and Ile-Ala-Gly-Gln-d-Arg. The substrates and the two products were separated on a reversed-phase column (Varian MCH-10) eluted as follows Initially the...

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

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....

Analysis Of Pen Inks

Forensic science ink analysis is one of the most traditional analytical fields. Currently, thin-layer and column chromatography as well as slab gel electrophoresis are used to investigate ink composition, but, recently, CZE has been tentatively applied, with encouraging results Fanali and Schudel, 1991 . Reversed-polarity separations were carried out in a silica-coated capillary 200 mm X 25 mm i.d. using 0.1 M ammonium acetate buffer pH 4.5 , with methanol 3 1 . Detection was by UV absorbance...

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...