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Immobilization of whole cells has also received attention since it has the advantage of not requiring the separation and puri cation of the active enzyme from the rest of the cell in order to produce the immobilized enzyme Also, in some instances the active enzyme is active only in the presence of other species (coenzymes, cofactors) The use of whole cells avoids the need to establish the identity of the other required species followed by their separation and incorporation in the immobilized enzyme Af nity chromatography, a chromatographic method of separation and puri cation, is an application of immobilized enzymes [Chaiken et al, 1984; Cuatrecasas and An nsen, 1971; Jakoby and Wilchek, 1974] An immobilized enzyme constitutes the adsorbent in a chromatography column.



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n=-(X)

When a mixture of biochemicals, such as a mixture of proteins, is passed through the column, a speci c interaction (often through covalent bonding) between the immobilized enzyme and one of the components of the mixture separates that component from the mixture Subsequent passage of the appropriate reagent through the column reverses the interaction and frees the desired component from the column Immobilized enzymes in the form of af nity chromatography, electrodes, and other sensing devices are of interest for analytical purposes (including immunoassays)..

(EI8)

9-14

y:<X)

The general scheme for utilizing a polymer substrate for synthetic purposes is shown in Fig. 9-2. A substrate S is covalently bonded to an appropriate polymer support to yield the polymer substrate (which is usually of the insoluble type). The polymer substrate P S





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,...,

Fig. 9-2 Scheme for utilization of a polymer substrate. After Mathur and Williams [1976] (by permission of Marcel Dekker, New York).

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is treated with a low-molecular-weight reagent (and/or catalyst) R to yield the polymerP S bound product 0 and spent reagent R0 . Filtration separates the polymer-bound product. An appropriate reaction is carried out to cleave the desired product S0 from the polymer. The spent polymer support is ltered off, leaving the crude product S0 in the ltrate. The puri ed product S0 is obtained from the ltrate after solvent evaporation and appropriate puri cation.

FigureF.3 A plot of the comb function (F.18).

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One of the most important applications of the polymer substrate technique is the solid-phase synthesis of polypeptides developed by Merri eld [Barany et al., 1988; Erickson and Merri eld, 1976; Kates and Abericio, 2000; Kent, 1988; Merri eld, 1978; 1985, 1988; Seward and Jakubke, 2002; Stewart, 1980]. Merri eld received the 1984 Nobel Prize in Chemistry for this achievement. Polypeptide synthesis is of interest for two reasons: (1) it is useful in con rming the structure of naturally occurring biological macromolecules and (2) it is a source of these substances as well as analogs that may show more desirable biological activity. Polypeptide synthesis involves the formation of an amide linkage between the amino and carboxyl groups of successive a-amino acid monomers. In reacting two different amino acids with each other, it is necessary to protect the amino group of one amino acid and protect the carboxyl of the other amino acid. This prevents self-reaction of each amino acid with itself and directs the synthesis in the desired cross-reaction route. There are two copolymerization routes and only one of these is the desired route. For example, alanylglycine (Eq. 9-72) and glycylalanine (Eq. 9-73) are possible from the cross-reaction of alanine and glycine. Synthesis of alanylglycine requires that the amine end of alanine and the carboxyl end of glycine be protected prior to reaction. Synthesis of glycylalanine requires that the amine and carboxyl ends of glycine and alanine, respectively, be protected.

9-73

- L < x < L which is reproduced periodically throughout the entire real line (see Figure F.3), if we now take the limit of vanishing , we obtain a periodic train of delta functions,

9-72

lim yf(x)

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