Immobilized carboxy

Very few attempts have been made to use soluUe polymer supports in the synthesis of peptides for extension of the carboxyl terminus. One example has been published by Royer and Anantharamaiah [33]. They used carboxymethyl polyfoxyeth ne) for the synthesis of peptides in aqueous phase with water-soluble coupling agents and immobilized carboxy-peptidase Y for cleavage of the protecting groups ... 

Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

Various pH sensors have been built with a fluorescent pH indicator (fluorescein, eosin Y, pyranine, 4-methylumbelliferone, SNARF, carboxy-SNAFL) immobilized at the tip of an optical fiber. The response of a pH sensor corresponds to the titration curve of the indicator, which has a sigmoidal shape with an inflection point for pH = pK , but it should be emphasized that the effective pKa value can be strongly influenced by the physical and chemical properties of the matrix in which the indicator is entrapped (or of the surface on which it is immobilized) without forgetting the dependence on temperature and ionic strength. In solution, the dynamic range is restricted to approximately two pH units, whereas it can be significantly extended (up to four units) when the indicator is immobilized in a microhetero-geneous microenvironment (e.g. a sol-gel matrix). 

A variable quantity of ZnO is added to the concentrated KOH solution, depending on the system characteristics required. ZnO can also act as a gassing suppressor. The electrolyte is immobilized generally using carboxy-methylcellulose, and a non-woven fabric separator made of natural or synthetic fibres resistant to the high pH is placed between the electrodes. 

Heparin is most tightly bound to a polymeric material when immobilized via covalent bonding. The heparin molecules contain hydroxy, carboxy, sulfate, and amino groups which are all suitable for this purpose. However, heparin is soluble in water and form-amide only and is insoluble in organic solvents which presents a significant restriction for covalent conjugation of heparin. 

The triazene linker can be used for the immobilization of aromatic diazonium salts, and therefore for aromatic amines, but not for aliphatic amines due to the instability of their diazonium salts. Cleavage of the linker can be achieved under mild acidic conditions to yield the benzylamine resin and the corresponding diazonium salt [136,145,146]. The main difference between the preparation of triazenes in solution and triazenes on solid support is the respective amine, namely bisben-zylamine and polymer-supported benzylamine 114. In solution, it was used in excess to quench unstable diazonium salts and force the reaction to completion. In the solid-phase approach it was immobilized and cannot be used in excess with respect to low loadings. A simple three-step procedure (Scheme 36) starting from benzylamine resin 114 via carboxylate 115 led to the successful preparation of ester resins 116 in essentially higher loadings. Treatment of resin 114 with 4-carboxy-benzene diazonium tetrafluoroborate yielded benzoic acid resin 115. 

Like many other useful discoveries, enzyme immobilization by cross-linking was actually an unintended by-product of another research project. In 1964, Florante Quiocho and Frederic Richards at Yale university cross-linked crystals of carboxy-peptidase-A with glutaraldehyde (pentane-1,5-dial), hoping to get stable crystals for X-ray diffraction studies. They noted that these cross-linked enzyme crystals (now... 

In 1996, Gauglitz and coworkers coated surfaces with various amino-and carboxy-substituted polymers [198], The polymers tested were branched poly-(ethyleneimine), a,co-amino-functionalized PEG, chitosan, poly(acrylamide-co-acrylic acid) and an amino-modified dextran. The amino-substituted polymers were immobilized on glass by first immobilizing an aminosilane, followed by succinic anhydride/A-hydroxysuccinimide linker chemistry. Poly(acrylamide-co-acrylic acid) was directly coupled to an aminosilanized surface. When probed with 1 mg mL 1 ovalbumin solution, nonspecific adsorption was lowest for the dextran derivative. Notably, nonspecific adsorption increased in most cases when a hydrophobic hapten (atrazine) was coupled to the polymer-modified surface. 

The carboxy group of PAA (MW 1 080 000) is covalently modified with amino-linked ssDNA as probe and PDPH for self-assembly immobilization. The chemical structure of the polymer unit is shown in Fig. 8. The amounts of ssDNA and PDPH in the polymer were determined by absorption measurements to be equivalent to 1/1500 [molecule/monomerunit] and 1/93 [molecule/monomerunit],respectively. 

Chemical Attachment. The initial step in chemically attaching enzymes to electrodes involves an activation (derivatization) of the support to introduce appropriate functional groups, such as carboxy, phenol, and quinone-like structures. This step is critical and must be controlled rigorously since the greater the number of functional groups, the higher will be the amount of immobilized enzymes attched and the better the final electrode activity. The electrode surface may be activated by different ways ... 

By using a resonant mirror biosensor, the binding between YTX and PDEs from bovine brain was studied. The enzymes were immobilized over an aminosilae surface and the association curves after the addition of several YTX concentrations were checked. These curves follow a typical association profile that fit a pseudo-first-order kinetic equation. From these results the kinetic equilibrium dissociation constant (K ) for the PDE-YTX association was calculated. This value is 3.74 p,M YTX (Pazos et al. 2004). is dependent on YTX structure since it increases when 44 or 45 carbons (at C9 chain) group. A higher value, 7 p,M OH-YTX or 23 p,M carboxy-YTX, indicates a lower affinity of YTXs analogues by PDEs. 

Recombinant human interleukin-2 (rIL-2, previously known as T-cell growth factor) was expressed in E. coli (7). During the purification process of rIL-2 by reversed-phase HPLC, another higher molecular weight form of this protein was also isolated, known as HMW rIL-2 (8). The purification process of HMW rIL-2 involved two chromatography steps and utilized a Bakerbond Carboxy-Sulfon (CS) column. Approximately 2 nmol each of rIL-2 and HMW rIL-2 were immobilized using ProSpin cartridges for C-terminal sequence analysis. 

Protease-free preparation of CNTs can be obtained using an immobilized-metal-ion affinity chromatography (IMAC) step (Ros-setto et a/., 1992). This procedure is also useful for the purification of He, the 50 kDa carboxy-terminal part of the heavy chain of TeTx, which shows an identical retention time. However, IMAC-chromatography cannot be used for the purification of BoNT/D because this serotype is not retained. To obtain a protease-free BoNT/D preparation, an ion-exchange chromatography procedure was used (Schiavo and Montecucco, 1995). After freezing in liquid nitrogen, purified CNTs are stored at -80°C. 

---