Immobilization Methods
Enzyme immobilization methods are classified as chemical or physical. Chemical methods involve the formation of covalent bonds between functional groups on the Bianalytical Chemistry, by Susan R. Mikkelsen and Eduardo Corton ISBN 0-471-54447-7 Copyright 2004 John Wiley amp Sons, Inc. support material also called the matrix or the carrier and functional groups on the enzyme. Chemical methods are subclassified as either nonpolymerizing or cross-linking methods. Nonpolymerizing methods involve the...
Structural And Functional Properties Of Antibodies
When the red blood cells are removed from whole blood, and the resulting plasma is allowed to clot, the fibrinogen may be removed to yield serum. Serum contains a variety of proteins, some of which are called globulins because their solubility properties are different from the other serum proteins. Antibodies are a subclass of serum globulins that possess selective binding properties. Antibodies are also called immunoglobulins Ig . All immunoglobulins have a number of structural features in...
Theoretical Treatment Of Packedbed Enzyme Reactors3233
Packed-bed enzyme reactors, those employing enzymes immobilized onto a particulate phase that is subsequently packed into a column, may be characterized by their column capacity, C, and the degree of reaction P. The parameter C is defined in Eq. 4.23, where k is the decomposition rate constant for the enzyme-substrate complex either k2 or kcat , Et is the total number of moles of enzyme immobilized, and the value of b is a constant for a given reactor, and is equal to the ratio of reactor void...
Total Carbohydrate
Methods reported for the quantitation of reducing sugars, that is, those that possess a terminal aldehyde group, involve redox reactions with oxidizing agents such as Cu2 in the presence of BCA or Fe CN 63 . Nelson reagents alkaline copper sulfate and solutions of molybdate and arsenate in dilute sulfuric acid that reduce to molybdenum blue , or 2-cyanoacetamide. The Cu -BCA assay has been described earlier for protein quantitation, and the method used for quantitation of reducing carbohydrates...
Bioanalytical Chemistry
A JOHN WILEY amp SONS, INC., PUBLICATION Copyright 2004 by John Wiley amp Sons, Inc. All rights reserved. Published by John Wiley amp Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either...
Precipitation Reactions
Precipitation reactions are similar in principle to agglutination reactions, the difference being that the antigen is a soluble, molecular species rather than a suspended particle such as a bacterium or erythrocyte. At a certain Ab Ag ratio, the cross-linked polymeric network of antibody and antigen loses its solubility and precipitates. This is called the precipitin reaction.11 Figure 5.6 shows how the quantity of precipitin produced varies with the quantity of antigen added, for a fixed total...
Competitive Inhibition
Competitive inhibitors compete with the substrate for the enzyme's active site, but are not converted to products after they are bound. They block the active site from substrate, and their effectiveness is described by their inhibition constant, KJ, which is the dissociation constant of the enzyme-inhibitor complex k 3 k3 E S E S E P 2.38 Jn this model, the enzyme-inhibitor complex is completely inactive, but is in equilibrium with the active form of the enzyme. For a simple one-substrate...
Enzyme Units And Concentrations
Enzyme concentrations may be represented in an identical manner as with nonca-talytic species, such as is in molar units or as mass per unit volume. The information required for this calculation is minimal the percentage purity of the enzyme preparation, the molecular weight of the enzyme, and the mass and volume of the solution prepared. Enzyme concentrations are rarely represented in this way, however, because such values say nothing about the catalytic power of the solution prepared. The...
References
1. R. R. Alexander and J. M. Griffiths, Basic Biochemical Methods, Wiley-Liss, New York, 1993, pp. 29-31. 2. C. M. Stoscheck, Methods Enzymol. 182, 1990, 50-68, and references cited therein. 3. G. Legier, C. M. Muller-Platz, M. Mentges-Hettkamp, G. Pflieger, and E. J lich, Anal. Biochem. 150, 1985, 278-287. 4. R. J. Kessler and D. D. Fanestil, Anal. Biochem. 159, 1986, 138-142. 5. M. G. Redinbaugh and R. B. Turley, Anal. Biochem. 153, 1986, 267-271. 6. S. J. Compton and C. G. Jones, Anal....
Experimental Determination of MichaelisMenten Parameters
Equations 2.18 and 2.19 show that the two regions of analytical utility in the rate versus substrate concentration profile of an enzyme occur at S lt 0.1 Km for substrate quantitation and S gt 10 Km for enzyme quantitation . In developing a new assay, or in adapting an established assay to new conditions, it is thus important to establish the Km value of the enzyme. It is of practical importance to also establish Figure 2.7. Eadie-Hofstee plot for v measurements with varying S . Figure 2.7....
Comparison of Methods for the Determination of Km Values10
The precisions and accuracies of the four methods described above for the determination of Km values were compared statistically by first generating an error-free data set of v and S data, and then introducing different types of error to the v values to generate a total of 100 individual data sets. The error-free data set was generated using Eq. 2.17 with Vmax Km 1, and the seven individual values are given below in Table 2.3. Fifty data sets containing absolute errors were then generated by...
Immobilized Enzyme Reactors
The immobilization of enzymes onto particulate carriers that may be packed into a column the ''packed-bed'' reactor , such as a typical HPLC column, facilitates Figure 4.9. Diagram of an open enzyme reactor system. Figure 4.9. Diagram of an open enzyme reactor system. repetitive use of the enzyme and also allows the automation of enzymatic assays.28 Open-tubular reactors have also been constructed by covalently immobilizing an enzyme onto the inner wall of a nylon or polyethylene tube.29...
Problems 1
1. The enzyme alanopine dehydrogenase reversibly catalyzes the reaction shown below. It is present in bivalve molluscs, such as the periwinkle, that live in intertidal regions. These species survive exposure during low tides through valve closure, and their metabolism switches from the aerobic lactate dehydrogenase pathway to the anaerobic alanopine dehydrogenase pathway. Exposed mollusks accumulate alanopine in their tissues, and alanopine levels have been related to exposure times. NH CH CH3...
Suggested References 1
O. Zaborsky, Immobilized Enzymes, CRC Press, Cleveland, OH, 1973. L. B. Wingard, Jr., E. Katchalski-Katzir, and L. Goldstein, Eds., Applied Biochemistry and Bioengineering, Volume 1 Immobilized Enzyme Principles, Academic Press, New York, 1976. L. B. Wingard, Jr., E. Katchalski-Katzir, and L. Goldstein, Eds., Applied Biochemistry and Bioengineering, Volume 3 Analytical Applications of Immobilized Enzymes and Cells, Academic Press, New York, 1981. P. W. CarrandL. D. Bowers, Chemical Analysis,...
Properties Of Immobilized Enzymes
The behavior of immobilized enzymes differs from that of dissolved enzymes because of the effects of the support material, or matrix, as well as conformational changes in the enzyme that result from interactions with the support and covalent modification of amino acid residues. Properties observed to change significantly upon immobilization include specific activity, pH optimum, Km, selectivity, and stability.23 Physical immobilization methods, especially entrapment and encapsulation, yield...
Free Fatty Acids
Most methods for the quantitation of free fatty acids involve chemical derivatiza-tion followed by gas chromatography-mass spectrometry. However, a colorimetric method is available for the quantitation of long-chain C gt 10 free fatty acids in plasma, and is based on the color developed by cobalt soaps of free fatty acids dissolved in chloroform.31 The assay involves four solutions 1 chloroform heptane methanol 4 3 2 by volume 2 0.035 M HCl 3 the salt reagent, consisting of 8 mL of...
Nonpolymerizing Covalent Immobilization
Covalent immobilization methods rely on functional groups on both the enzyme and the support material for the formation of stable covalent bonds. For this reason, the choice of a support is crucial in that it determines the immobilization chemistry Figure 4.1. Enzyme immobilization methods. a Nonpolymerizing, b cross-linking, c adsorption, d entrapment, and e encapsulation. Figure 4.1. Enzyme immobilization methods. a Nonpolymerizing, b cross-linking, c adsorption, d entrapment, and e...
Diaminobenzoic Acid Method
Samples containing DNA are incubated in 1 N perchloric acid for 10 min. Under these conditions, DNA is depurinated, deoxyribose is released, and converted to o-hydroxylevulinylaldehyde. An equal volume of 1.3 M DABA dihydrochloride is TABLE 1.1. Detection Limits and Interferences for Protein Assay Methods TABLE 1.1. Detection Limits and Interferences for Protein Assay Methods Acidifiers, Cu chelating agents e.g., high phosphate concentration , Cu2 reducing agents e.g., reducing sugars, thiols ,...
Ferricyanide Method
This assay employs an alkaline solution of potassium ferricyanide 2.5 mg mL in phosphate buffer as the only reagent. A reducing sugar solution of 80 pL is combined with 20 pL of this reagent. The mixture is heated in a sealed tube to 100 C for 10 min. Following the addition of 300 pL of cold water, absorbance is measured at 237 nm, which is the wavelength of maximum absorbance for ferrocyanide. This method allows the quantitation of mannose over the 1-25-nmol 1.3-30-pg mL range of reducing...
Electrochemical Detection
3.5.2.1. Amperometry. Amperometric methods measure the current produced at a working electrode in response to an applied potential. Amperometric enzyme assays rely on the production of an oxidizable or reducible species from an enzyme-catalyzed reaction. The applied potential is extreme enough to completely oxidize at positive potentials or reduce at negative potentials any analyte that contacts the working electrode. In stirred or unstirred solutions, the current produced under such...
OneSubstrate TwoProduct Enzyme Kinetics
Hydrolytic enzymes that release two products in a defined sequence require a slightly more complicated model for their kinetic behavior. These enzymes pass through two intermediate stages in the catalytic cycle, E S and E S', and the formation of products involves two steps with associated rate constants k2 and k3 E S E S E S' Pi P2 Pi 2.24 The rate-determining step is assumed to involve the release of the first product P1 and the conversion of E S into E S'. Application of the steady-state...
NinhydrinBased Assay8
This method is based on the quantitation of total amino acids following acid hydrolysis of proteins present in tissue samples. Microtiter plates are used in this assay. Tissue samples 10 mg are first hydrolyzed in 500 mL of 6 M HCl at 100 C for 24 h to liberate ammonium. The samples are then lyophilized chilled and evaporated , and the residue, containing ammonium chloride, is dissolved in a known volume of water. Ninhydrin reagent, containing ninhydrin, ethylene glycol, acetate buffer, and...
Problems
1. Total protein in an unknown sample was estimated using the Lowry and Bradford assays. Results were 33 2 mg mL from the Lowry assay and 21 1 mg mL from the Bradford assay, using stock solutions of BSA as standards in each assay. The unknown sample was then thoroughly oxygenated, and the assays were repeated. The Lowry method yielded 22 1 mg mL, while the Bradford results did not change. Why did the results of the Lowry assay decrease by 33 2. The Lowry, Smith, and Bradford assays provide an...
Enzyme Nomenclature
Enzyme names apply to a single catalytic entity, rather than to a series of individually catalyzed reactions. Names are related to the function of the enzyme, in particular, to the type of reaction catalyzed. This convention implies that one name may, in fact, designate a family of enzymes that are slightly different from each other yet still catalyze the same reaction. For example, lactate dehydrogenase LDH has five such isoenzymes in humans. LDH catalyzes the oxidation of L-lactic acid by...
Enzyme Commission Numbers
The ultimate identification of a particular enzyme is possible through its Enzyme Commission E.C. number.2 The assignment of E.C. numbers is described in guidelines set out by the International Union of Biochemistry, and follows the format E.C. w.x.y.z, where numerical values are substituted for w, x, y and z. The value of w is always between 1 and 6, and indicates one of six main divisions values of x indicate the subclassification, and are often related to either the prosthetic group or the...
Prosthetic Group Chemistry
Enzymes are biological catalysts that facilitate the conversion of substrates into products by providing favorable conditions that lower the activation energy of the reaction. Substrate s - gt Product s 2.1 An enzyme may be a protein or a glycoprotein, and consists of at least one polypeptide moiety. The regions of the enzyme that are directly involved in the catalytic process are called the active sites. An enzyme may have one or more groups that are essential for catalytic activity associated...
Other Protein Quantitation Methods
The method of nitration has been reported for protein quantitation. In this method, aromatic amino acid residues are treated with nitric acid, and the products of nitration absorb at 358 nm. In the case of tyrosine, nitration occurs at the 3-position of the aromatic ring, producing 3-nitrotyrosine. This method has been compared to the Smith and Bradford assays using BSA standards, and practically identical results were obtained, as shown in Figure 1.5. The detection limit of this assay is 5 mg,...
Other Fluorometric Methods
Sensitive fluorometric assays for double-stranded native DNA in tissue extracts are based on the noncovalent interaction of intercalating dyes Fig. 1.7 such as ethi-dium bromide, 4',6'-diamidino-2-phenylindole DAPI , or 1-methyl-4-piperazyl benzimidazol trihydrochloride Hoechst 33258 , with double-stranded DNA in neutral, aqueous solutions. When free in aqueous solution, these dyes exhibit limited fluorescence, but upon binding DNA, their fluorescence increases markedly. Ethidium binds to RNA...
Purpald Assay for Bacterial Polysaccharides
Polysaccharides containing substituted or unsubstituted glycols in residues such as glycerol, ribitol, arabinitol, furanosyl galactose, and sialic acid may be quantitated by reaction with sodium periodate, follow by reaction with the purpald reagent, as shown in Figure 1.8. The formaldehyde produced in the first step reacts with the purpald reagent, and the product of this reaction is further oxidized by periodate to form a purple product with an absorbance maximum at 550 nm. The limit of...
Why Does Rna Absorbs Maximum At 660 Nm With Orcinol
The only method used to quantitate ribonucleic acid is the orcinol assay.20 With this method, RNA is depurinated in concentrated HCl and the resulting ribosephos-phates are dephosphorylated and dehydrated to produce furfural Eq. 1.5 . Furfural then reacts with orcinol in the presence of Fe3 to yield colored condensation products, as shown in Eq. 1.4, which together possess an absorption maximum at 660 nm. The DNA also undergoes a limited reaction under these conditions, and yields 10 of the...
Diphenylamine Method
The DPA method is based on the colorimetric measurement of products formed by reaction of o-hydroxylevulinylaldehyde from the deoxyribose released after depurination of DNA with diphenylamine in 1 M perchloric acid.14-16 The reaction yields a mixture of products, such as that shown in Eq. 1.4,17 which together have an absorbance maximum at 595 nm. This method has a similar dynamic range to the DABA assay with colorimetric measurement. Interferences include proteins, lipids, saccharides, and...
PhenolSulfuric Acid Method
In concentrated sulfuric acid, polysaccharides are hydrolyzed to their constituent monosaccharides, which are dehydrated to reactive intermediates. In the presence of phenol, these intermediates form yellow products, such as that shown in Eq. 1.6,28 with a combined maximal absorbance at 492 nm. The phenol-sulfuric acid method has been adapted for use with 96-well micro-titer plates. Carbohydrate samples are combined with an equal volume of 5 w v phenol in the wells of a microtiter plate, the...
Nomograph E. Adams
The objective of many bioassay methods is to selectively quantitate a single biomo-lecule, such as a particular enzyme or antibody, or to determine the presence or absence of a known DNA sequence in an unknown sample. Methods for these very selective assays will be considered in later chapters. When faced with a true unknown, for example, during the isolation or purification of a biomolecule, it can be important to characterize the unknown matrix, or the components in the unknown solution that...
Smith BCA Method
The Smith total protein assay is also based on the initial complexation of copper II with peptides under alkaline conditions, with reduction to copper I . The ligand BCA is then added in excess, and the purple color 562-nm peak absorbance develops upon 2 1 binding of BCA with Cu Fig. 1.2 . Figure 1.2. Complex formed upon reaction of bicinchoninic acid with Cu Figure 1.2. Complex formed upon reaction of bicinchoninic acid with Cu The Smith assay takes 1 h, and requires a minimum volume of 500...
Bradford Method
The Bradford method is based on the noncovalent binding of the anionic form of the dye Coomassie Blue G-250 with protein.6 The dye reacts chiefly with arginine residues, which have a positively charged side chain, and slight interactions have also been observed with basic residues histidine and lysine and aromatic residues tyrosine, tryptophan, and phenylalanine . In the absence of protein, the dye reagent is a pale red, and upon binding to protein, a blue color is generated with an absor-bance...
Lowry Method
The Lowry, or enhanced alkaline copper method, begins with the addition of an alkaline solution of Cu2 to the sample. The copper forms a complex with nitrogen atoms in the peptide bonds of proteins under these conditions, and is reduced to Cu . The Cu , along with the R groups of tyrosine, tryptophan, and cysteine residues of the protein then react with the added Folin-Ciocalteau reagent, which contains sodium tungstate, sodium molybdate, phosphoric acid, and HCl W6 Mo6 . During this reaction,...