Peroxidase protein cross-linking

Matheis G, Whitaker JR (1984) Peroxidase-catalyzed cross linking of proteins. J Prot Chem 3 35-48... 

FIGURE 9.11 Relative mass change as a result of water absorption and loss in a solid piece of recombinant resilin prepared from a 20% protein solution in phosphate-buffered sahne (PBS), cross-linked using peroxidase (see [29] supplementary material). The fuUy swollen sample is designated 100. (Data courtesy of Shekibi, Y., Naim, K., Bastow, T.J., and HiU, A.J., The states of water in a protein based hydrogel. Internal CSIRO... 

Figure 15.9 The results of capture ELISA on native RNase A and formalin-treated RNase A. Right panel, native RNase A (curve 1) and unfractionated formalin-treated RNase A (curve 2). Left panel, individual fractions of formalin-treated RNase A monomer (curve 3), dimmer (curve 4), trimer (curve 5), tetramer (curve 6), and a mixture of oligomers with >5 cross-linked proteins (curve 7). The ELISA plate wells were coated with monoclonal antibody against bovine pancreatic RNase A (1 pg/mL) overnight at 4°C and then blocked with bovine serum albumin. The wells were incubated for lh at 37°C in the presence of various concentrations of antigen in lOOpL of PBS. After washing, each plate well received a 1 4000 dilution of horseradish peroxidase conjugated rabbit polyclonal anti-RNase A antibody followed by incubation at ambient temperature for lh. After washing, detection was achieved using a mixture of 2,2,-azino-di-(3-ethylbenzthiazoline-6-sulphonate) and hydrogen peroxide. Absorbance was monitored at 405 nm. See Rait etal.11 for details.

Thus, glycoproteins such as horseradish peroxidase, glucose oxidase, or most antibody molecules can be activated for conjugation by brief treatment with periodate. Cross-linking with an amine-containing protein takes place under alkaline pH conditions through the formation of Schiff base intermediates. These relatively labile intermediates can be stabilized by reduction to a secondary amine linkage with sodium cyanoborohydride (Fig. 303). 

Peroxidases oxidize tyrosines, both as a free amino acid and as a residue in peptides and proteins. When proteins are treated with HRP in the presence of hydrogen peroxide, protein dimers are obtained through the coupling of tyrosyl radicals. HRP can also be used for cross-linking of proteins with polysaccharides [35]. In this case, coupling occurs between a tyrosyl radical in the protein and a radical species on the saccharide ... 

The accumulation of hydrogen peroxidase affects many intracellular processes and results in hemolysis. These include the cross-linking of membrane proteins hemoglobin denaturation (manifest as Heinz body formation), which in turn affects the physical properties of the erythrocyte and lipid peroxidation, which may affect the cell membrane to cause direct hemolysis (Fig. 11-8). The resultant damage leads to a mixture of intravascular hemolysis and extravascu-lar hemolysis (by which hemolysis occurs in the reticuloendothelial system). In acute hemolytic episodes, the clinical picture is of predominantly intravascular hemolysis, while predominantly extravascular hemolysis is seen in patients with chronic hemolysis. 

The cross-reactivity of polyclonal antibodies raised to an atrazine-protein conjugate (linked at the chlorine group) was evaluated, using a peroxidase-atrazine labeled species linked in the same manner, in order to determine the reliability of a newly available competitive immunoassay kit for environmental atrazine monitoring.28 A wide variety of similar compounds (Fig. 6.14) were screened, and the concentrations given beside the compounds indicate the apparent concentration of atrazine that would be erroneously recorded from the responses obtained to 1-pg/ mL concentrations of each of these interferents. 

In 1994, Fawell et al. marked a second milestone, that the Tat protein can mediate the internalization of a heterologous protein into cells by chemical conjugation (Table 1) [20]. They chemically cross-linked Tat peptides (residues 1-72 or 37-72) to p-galactosidase, horseradish peroxidase, RNase A, and domain III of pseudomonas exotoxin A (PE) and monitored their uptake. Interestingly, all the cells in the culture dish were transduced with the Tat protein. In addition, the internalization was achieved in all cell types tested, such as HeLa, COS-1, CHO, H9, NIH3T3, primary human keratinocytes, and... 

Two types of enzymes were used to introduce cross-links in protein films peroxidase and transglutaminase. A treatment by horseradish peroxidase in the preparation of films of thermally denaturated soy proteins reduced the elongation at elastic limit and increased the tensile strength. ... 

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