Biochemical applications

A variety of methods are available to detect proteins separated by electrophoresis or to measure the concentration of total protein in a solution. These methods are normally based on the binding of a dye to one of the amino acids in protein, or a color reaction with an amino acid side chain. The most commonly used stains for protein detection on gels are Coomassie Brilliant Blue (98) and silver stain (99,100). These methods detect any protein residues, either in solution or on an electrophoresis gel. Their main requirement is sensitivity, not specificity. New, more sensitive dyes are being developed for the proteomic analysis of protein structure and sequence, for example Ruby Red (101). 

The most common method of radiolabeling employs Chloramine-T, the sodium salt of the N-monochloro derivative of p-toluenesulfonamide (Fig. 17). It breaks down slowly in aqueous solution to hypochlorous acid and is used as a mild oxidizing agent in radioiodination reactions. Other oxidation reagents used in radioiodination include 

In the presence of chloramine-T under mildly alkaline conditions (pH 7.5), Nal is oxidized to form cationic iodine I +. At this pH the tyrosine will be only slightly anionic as the pK of the phenolic side 

The enzymes commonly used as labels in ELISA and other immunochemical reactions include horse radish peroxidase (HRP) and alkaline phosphatase (AP). The enzyme can be covalently coupled to the antibody using glutaraldehyde conjugation to reactive amino groups on the enzyme (lysines) in a phosphate buffered aqueous solution at neutral pH, as shown in Fig. 19 (103). Alternatively, carbohydrates present in the immunoglobulin structure can be cleaved by periodate treatment (see Fig. 20) and bound to free amino groups on the enzyme through a Schiff base reaction (103). 

As shown in Fig. 21, in a direct ELISA the unlabeled antigen (a range of standard antigen concentrations or unknown samples) is attached to the solid phase. Enzyme-conjugated (labeled) primary antibody is then added. After incubation and washing of the plate 

The remaining solution, which has been termed PPL, withstands heating at 60°C for 10 hours and is still qualified as a substitute for plasma as far as oncotic and nutritive effects are concerned. 

Stahmann et al. (S12) studied the antigenic action of synthetic polypeptides with ImEl. The latter were composed of leucine, phenylalanine, glutamic acid, and lysine these polypeptides were coupled to a homologous protein, the serum albumin of the rabbit. From the pattern of the precipitate lines, it followed that antigenicity decreased in the following order leucine phenylalanine glutamic acid lysine. 

Vaux St. Cyr (VI) studied perchloric acid extracts of normal and of pathological sera. All of them contained seromucoid acid, and the latter sera contained in addition y-globulin, PsA-globulin, and albumin, but in variable amounts. 

2 mC/mg. The self-decomposition of compounds labeled with radioactive isotopes, leading to the disintegration of unstable atomic nuclei, has been described by Bayly and Weigel (B2). Further studies lead to a better knowledge of the properdin-zymosan and properdin-inulin complex (S3). 

Bargob, J., Cleve, H., and Hartmann, F., Immunelektrophoretische Serum-untersuchungen bei Lebererkrankungen. Deut. Arch. klin. Med. 204, 708-720 (1958). 

TABLE 7.6 Characteristics of main chain IR bands of proteins 

Vibration mode (cm ) a Helix Dichroism P Sheet Dichroism  

Note The amide I band at 1650 cm involves C=0 stretching (80%), C—N stretching (10%) and in-plane N—bending (10%) contributions. The amide II band at 1550 cmT involves a mixture of C—N stretching (40%) and in-plane N—bending (60%) contributions. Dichroism refers to polarization along the long axis. 

TABLE 7.7 Characteristic vibrations localized in the side chains of proteins 

However, the amide II bands are strongly overlapped by bands originating from amino acid side chains vibrations. 

Small organic molecules can be labeled with C-carbon by chemical or enz5miatic incorporation of into the molecular 

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P. R. Carey, ed.. Biochemical Applications of Raman and Resonance Raman Spectroscopies, Acedemic Press, Inc., New York, 1982. 

J. R. Lakowicz, ed.. Topics in Fluorescence Spectroscopy, Vols. 1—4 (Techniques-, Principles-, Biochemical Applications-, and Probe Design and Chemical Sensing), Plenum Press, New York, 1991—1994. 

Several features of this treatment are of interest. Compare the denominators of Eqs. (3-147) and (3-149) Miller has pointed out that the form of Eq. (3-147) is usually seen in chemical applications of the steady-state approximation, whereas the form of Eq. (3-149) appears in biochemical applications. The difference arises from the manner in which one uses the mass balance expressions, and this depends upon the type of system being studied and the information available. 

As a consequence of the high pressures that must be tolerated, LC sample valves are usually made from stainless steel. The exception to the use of stainless steel will arise in biochemical applications where the materials of construction may need to be bio-compatible. In such cases the valves may be made from titanium or some other appropriate bio-compatible material. 

Glasgow LA, Hua J, Yiin TY, Erickson LE (1992) Experimental studies of interfacial phenomena in sparged reactors. In Tatterson GB, Calabrease RV (eds) Process mixing chemical biochemical applications. AIChE Syposium Series 286 Garcia-Briones MA, Brodkey RS, Chalmers JJ (1994) Chem Eng Sci 49 2301 Boulton-Stone JM, Blake JR (1993) J Fluid Mech 254 437... 

Carey PR (1982) Biochemical applications of Raman and resonance Raman spectroscopies. Academic, New York... 

Since further reviewing pTAS could easily fill a separate book and is mostly concerned with biochemical applications, it was excluded from this book. Therefore, the description of these applications is beyond the scope of this chapter. The reader is referred to original reviews [31, 32]. 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

Kaetsu, Radiation Synthesis of Polymeric Materials for Biomedical and Biochemical Applications. VoL 105, pp. 81 -98. 

Wakselman, M. 1,4 and 1,6-Eliminations from hydroxy- and amino-substituted benzyl systems chemical and biochemical applications. Nouv J. Chim. 1983, 7, 439 147. 

Ingram, D. J. E. Biological and biochemical applications of electron spin resonance. London Adam Hilger 1969. 

D.J.E. Ingram, Biological and Biochemical Applications of ESR, Adam Hilger, London, 1969. 

Brecht A., Gauglitz G. Recent developments in optical transducers for chemical or biochemical applications, Sensors and Actuators B 1997 38 1-7. 

D. Wallace, Ink-jet based fluid microdispensing in biochemical applications. Nucl. Med. Biol. 21, 6-9... 

Poliakoff, M. Turner, J.J. "Chemical and Biochemical Applications of Lasers", Moore, C.B. Ed Academic Press ... 

Polymer-based microreactor systems [e.g., made of poly(dimethyl-siloxane) (PDMS)], with inner volumes in the nanoliter to microliter range (Hansen et al. 2006), are relatively inexpensive and easy to produce. Many solvents used for organic transformations are not compatible with the polymers that show limited mechanical stability and low thermal conductivity. Thus the application of these reactors is mostly restricted to aqueous chemistry at atmospheric pressure and temperatures for biochemical applications (Hansen et al. 2006 Wang et al. 2006 Duan et al. 2006). 

When addressing problems in computational chemistry, the choice of computational scheme depends on the applicability of the method (i.e. the types of atoms and/or molecules, and the type of property, that can be treated satisfactorily) and the size of the system to be investigated. In biochemical applications the method of choice - if we are interested in the dynamics and effects of temperature on an entire protein with, say, 10,000 atoms - will be to run a classical molecular dynamics (MD) simulation. The key problem then becomes that of choosing a relevant force field in which the different atomic interactions are described. If, on the other hand, we are interested in electronic and/or spectroscopic properties or explicit bond breaking and bond formation in an enzymatic active site, we must resort to a quantum chemical methodology in which electrons are treated explicitly. These phenomena are usually highly localized, and thus only involve a small number of chemical groups compared with the complete macromolecule. 

Another approach is that of including dynamics in the calculations. A dynamical formalism of DFT was first developed by Car and Parrinello [31], and has been employed in a wide range of areas, e.g. solvation problems, reactions on surfaces, solid-state interactions, and a variety of biochemical applications. In CP-MD one normally uses a plane wave basis to reduce the computational requirements and enable easy implementation of periodic boundary conditions. Nonetheless, CP-MD simulations are rather costly, and are normally not applied to systems larger than, say, 1-200 atoms, and over relatively short time frames. 

Sastry, S.D., Buck, K.T., Janak, J., Dressier, M. Preti, G. (1980) Volatiles emitted by humans. In G.R. Waller and O.C.Dermer (Eds.), Biochemical Applications of Mass Spectrometry, First Supplementary Volume. John Wiley, New York, pp. 1086-1129. 

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