Enzymatic techniques

Oxime carbamates are not directly amenable to gas chromatography (GC) because of their high thermal instability, which often leads to their breakdown at the injection port or in the column during analysis. Analysis of oxime carbamates by GC with sulfur detection or flame photometric detection involves oxidation of the intact insecticides or alkaline hydrolysis to form the more volatile but stable oxime compound. Enzymatic techniques have been reported for the analysis of these compounds. Enzyme-linked immunosorbent assay (ELISA) has been used to determine aldicarb and its sulfone and sulfoxide metabolites and methomyl in water, soil, and sediment samples. 

If an individual nucleotide is modified in the appropriate way, various enzymatic techniques can be used to polymerize the derivative into an existing oligonucleotide molecule. Alternatively, nucleotide polymers can be treated with chemical activators that can facilitate the attachment of a label at particular reactive sites. Thus, there are two main approaches to modifying DNA or RNA molecules enzymatic or chemical. Both procedures can produce highly active conjugates for sensitive assays to quantify or localize the binding of an oligo probe to its complementary strand in a complex mixture. 

Enzymatic techniques can employ a variety of DNA or RNA polymerases to add controlled amounts of modified nucleotides to an existing stand. However, the most common procedures utilize either DNA polymerase I or terminal deoxynucleotide transferase. The polymerase is used with a template to add modified nucleoside triphosphates to the end of a DNA molecule or to various sites within the middle of a sequence. The terminal transferase can add modified monomers to the 3 end of a chain without a template. 

The Polymerase Chain Reaction. In the past, a major drawback of hybridization assays was their need for relatively large amounts of sample DNA to compensate for their low sensitivity. This problem has been surmounted in recent years by the development of powerful enzymatic techniques that can exponentially replicate specific DNA sequences in the test tube. With these techniques it is now possible to analyze vanishingly small samples that initially contain fewer than 10 copies of the sequence of interest. The new methods take advantage of the chemical properties of nucleic acids and of highly specialized enzymes that can repair and replicate DNA in vitro. 

If cellular localization of the antigen-antibody complex is not required, enzyme immunolabeling can be performed on cells adherent to a microtiter plate, and the color change resulting from the enzymatic reaction can be detected as a change in absorbance with an automatic plate reader (see Chapter 28). Biotinylation of antibodies and the use of the avidin-biotin complex has further extended the versatility and sensitivity of the enzymatic techniques (see Chapters 7 and 25-27). Most recently, the principles behind these techniques have been applied in combination with in situ hybridization techniques. Using nucleic acid-antibody complexes as probes, specific DNA or RNA sequences can be localized (see Chapters 46 9). 

Guesdon, J. L., Temynck, T., and Avrameas, S. (1979) The use of avidin-biotin interaction in immuno-enzymatic techniques. / Histochem. Cytochem. 27,1131-1139. 

The objectives of the studies reported herein were to (a) compare the effects of a series of phenolic acids, coumarins, and flavonoids on whole chain electron transport and phosphorylation in Isolated plant chloroplasts and mitochondria and (b) identify specific sites of inhibition with polarographic and enzymatic techniques. Exploratory studies were conducted with the 20 compounds listed in Table I. The three glycosides are shown indented below the corresponding aglycones. Detailed studies were conducted with the six compounds, one representative member from each chemical family, designated with an asterisk. 

Enzymatic techniques have also been employed in the analysis of these compounds. The toxicity of carbamate insecticides is due to the inhibition of the enzyme acetylcholine esterase, so the determination of these compounds can be achieved by enzyme inhibition (2,83,119), bioassay (118,167), or enzyme-linked immunosorbent assay (ELISA) (168-171). In the detection of carbamates by fluorimetric enzyme inhibition, the effluent from a reversed-phase chromatographic column was incubated with cholinesterase, which was introduced via a postcolumn reagent delivery pump. Then, the resulting partially inhibited cholinesterase was reacted with N-methyl indoyl acetate to produce a fluorophore and a reduction in the baseline fluorescence (172). 

A similar semisynthetic method involves pronase E cleavage of leupeptin dibutyl acetal.157 Leupeptin dibutyl acetal has been prepared, cleaved by pronase E, and depro-tected as in the case of thermolysin, except pronase E cleaves the Leu-Arg bond instead of the Leu-Leu bond, thus affording H-Arg[CH(OBu)2]. This enzymatic technique was applied for the semisynthesis of Z-Val-Pro-Arg-Hj57 Again, the peptide dibutyl acetals can be treated with acid to afford the aldehydic leupeptin analogues with good yields and stereochemical conservation. 

Routinely, common chemical and enzymatic techniques are used to obtain protein fragments. Unfortunately, when enzymatic digestion techniques and nanograms quantities of proteins are used, the method become ineffective due to dilution and reduced enzymatic activity. An alternative approach to overcome this problem is the use of proteolytic enzymes immobilized to a solid support and a small-bore reactor column. Using trypsin immobilized to agarose, tryptic digests of less than 100 ng of protein can be reproducible obtained (49). 

Enzymatic Hydrolysis. Saccharification of wood polysaccharides to sugars can be accomplished by enzymatic techniques instead of acid hydrolysis. The U.S. Army Natick Laboratories developed a method for conversion of cellulose to glucose with a cellulose enzyme from an active strain of the fungus Trichoderma viride. However, extensive pretreatment of wood is necessary before sufficient enzymatic hydrolysis will take place. 

The Gulf Oil Company developed a method called simultaneous saccharification and fermentation (SSF) for the enzymatic conversion of waste paper to ethanol.38 In this process the cellulose is enzymatically hydrolyzed and the glucose yeast-fermented in one operation. This modification, along with improved enzyme production and performance, has made the enzymatic technique more economically viable for the conversion of waste paper to ethanol. The process was donated to the University of Arkansas for further development. 

The mechanism of penicillin biosynthesis from the Arnstein tripeptide, 8-(L-a-aminoadipoyl)-L-cysteinyl-D-valine (ACV), has been extensively studied and reviewed by many chemists. Most of the biosynthetic mechanisms have been ascertained by Baldwin and Bradley using an excellent enzymatic technique.55 However, the first step in the biosynthesis of penicillin, conversion of the Arnstein tripeptide to a cis-P-lactam intermediate, is still a fascinating mechanistic problem. Although Baldwin et al. recently proposed a mechanism involving an iron-bound thioaldehyde formation route via a Pummerer-type cyclization, the intermediate for this mechanism has not been identified. The mechanism of selective formation of the cw-P-lactam ring is still also unknown (Fig. 39).56 These types of biomimetic reactions have been chemically studied. An example of an unsuccessful intramolecular Pummerer cyclization of the sulfoxide involving a cysteine moiety under standard Pummerer conditions was reported by Wolfe et al.57 Although Kaneko reported the conversion of the very simple 3-phenylsulfinyl propionamide into a P-lactam with TMSOTf/triethylamine,58 a successful biomimetic synthesis of... 

The selective N-terminal deprotection of 0-glycopeptides by means of an enzymatic technique was achieved using the p-phenylacetoxybenzyloxycarbonyl... 

Chemical synthesis of glycopeptides which carry large oligosaccharide residues is often demanding and time-consuming. In such cases, use of enzymatic techniques for the elaboration of existing,... 

A Review of Enzymatic Techniques Used for Pesticide Residue Analysis... 

A review is presented on the use of enzymatic techniques for pesticide residue analysis. Anticholinesterase and anti-carboxylesterase procedures, which comprise the majority of such techniques, are described with reference to the theory behind their use, the different methods of assay, their limits of detection, and their advantages and disadvantages. Other enzymatic techniques are also discussed. 

T he fact that many pesticides inhibit enzymes in vitro has led to the introduction of various analytical methods for the detection and estimation of pesticide residues. This paper wiU not describe in detail all the enzymatic techniques used for pesticide residue analysis but rather will attempt to categorize and describe briefly the main types with reference to the theory behind their use, their practical application, and associated problems. This will be followed by a brief discussion on the advantages and disadvantages of these techniques. 

Small DNA segments can be synthesized in the laboratory, and commercial instruments are available for automating the work. Sequencing of DNA can be carried out either by the Maxam-Gilbert method, which uses chemical techniques, or by the Sanger dideoxy method, which uses enzymatic techniques. Small amounts of DNA can be amplified by factors of 10 using the polymerase chain reaction (PCR). 

Jarvie DR, Heyworth R, Simpson D. Plasma salicylate analysis A comparison of colorimetric, HPLC and enzymatic techniques. Ann Clin Biochem 1987 24 364-73. 

Carbohydrate analysis is much simpler in such samples. Pulsed amperometry is a very sensitive detection method and beverages to be analyzed can often be strongly diluted, so interferences caused by the matrix are usually not observed. To determine sorbitol in apple juice, for example, apple juice is simply diluted 1 1,500 with de-ionized water and injected directly (Fig. 8-65). Comparison measurements with enzymatic techniques... 

Table 8-8. Comparison of the results obtained with IC and enzymatic techniques for carbohydrate analysis in apple juice (taken from [57]).

The addition of a higher molecular weight acid such as capric acid, is beneficial, and under these conditions high yields of propionates are obtained, presumably via a transesterification mechanism. Using this enzymatic technique it is possible to carry out relatively large scale preparative syntheses of natural flavor-active esters, using natural raw materials as substrates. 

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