Crosslinking site-directed

In our study, the effect of moisture over the nonneutral pH range of 3-11, direct sunlight, ozone at a concentration level of 6000 ppm, and the effects of loading stresses, were investigated for the three commercial sealants. A characteristic variation of crosslink density for the typical silicone sealants is shown in Fig. 29. This figure depicts the results for the coupons exposed to moisture and sunlight. Initially upon exposure, the crosslink density of the sealants exhibit an increase due to the availability of residual uncurred crosslink sites... 

Fancy, D.A., and Kodadek, T. (1997) Site-directed oxidative protein crosslinking. Tetrahedron 53, 11953-11960. 

Zecherle, G.N. (1990) The ribosomal location and conformation of Escherichia coli protein L7/L12 studied by cysteine site directed mutagenesis and crosslinking. Doctoral Dissertation. University of California at Davis. 

Recent structural studies and electron transfer kinetic experiments focus on structures in which a site-specific covalent crosslink between cytochrome c and cytochrome c peroxidase subunits exists. One of these used site-directed mutagenesis to form a disulfide bond between a V197C mutant CcP and an A81C... 

Chemical modification of surface residues of HRP is one method which may offer some improvement in thermal or long-term stability of the enzyme. The -amino groups of the six surface Lys residues can be modified by reaction with carboxylic anhydrides and picryl sulfonic acid (296). In this example the number of sites modified was found to be more significant than the chemical nature of the modification, at least as a criterion for improved stability. Other methods explored include the use of bifunctional crosslinking reagents to couple surface sites on the enzyme (297). Future developments are likely to be concerned with the selection of site-directed mutants of HRP C that show enhanced thermal stability. Dramatic increases in thermal stability of up to 190-fold have been reported recently for mutants of Coprinus cinereus peroxidase (CIP) generated using a directed evolution approach (298). 

Elasticity measurements already have been used to detect the presence of crosslinking proteins in cytoplasmic extracts(24). If preformed actin chains are employed, assessment of the rheological state of a network directly provides information about the parameters of crosslinking. Moreover, chain growth can bg studied, although indirectly, through the effects of Cq, Uq, on the concentration of crosslinking sites Sy. 

Selective bond rupture at entanglement points, or other such sites of stress concentration, could magnify the effect of a chain scission in the presence of an external stress, but it seems unlikely that this is occurring since the sol-gel data actually indicated a (slightly) lower ratio of scissions to crosslinks with an imposed stress. It also is difficult to visualize how the formation of free radicals, scissions, and crosslinks could directly cause the radiation expansion noted under no stress. Therefore, the mechanism of accelerated creep is probably not caused by the formation and reaction of macromolecular free radicals in the polymer specimens. 

Figure 5-32 (A) A three-dimensional computer graphics model proposed by Brimacombe et a/.3 11 for the single chain of E. coli 16S ribosomal RNA. The helices are depicted as cylinders, which are all connected. The small dark squares denote the positions of artificially formed RNA-protein crosslinks, marked with the appropriate protein number. For proteins exhibiting more than one crosslink site (e.g., SI7), the sites are denoted A or B, in each case A being the site nearer to the 5 terminus of the 16S RNA. (B) Stereoscopic view of tentative atomic model of 16S RNA in the 30S ribosomal subunit. The viewing direction is different from that in (A). From Mueller and Brimacombe.342 Courtesy of Richard Brimacombe.

Fig. 104 Space-filling rendition of an epoxy-resin-like structure (grey), without crosslinks, showing the placement of the antiplasticiser (black) within the structure. Only the two chains directly above and below the antiplasticiser are shown in the figure. The labelled atoms are indicated by open circles. The antiplasticiser 13C-carboxyl carbon is 4.9 A from an amine 15N (with no preference for free or crosslink sites) and 6.7 A from a quaternary carbon of the isopropylidene moiety directly bonded to two C2H3 groups. The intramolecular distance from the quaternary carbon to the amine 15N is approximately 10 A (from [71])...

Despite these limitations, one can qualitatively conclude from the results depicted in Figure 1 that crosslinking increases with conversion. Furthermore, the relative rate of crosslinking, i.e., formation of crosslink sites as a function of styrene conversion, probably also increases with conversion. However, the second statement has yet to be proved in special experiments in which the formation of network sites is measured directly. 

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