Crosslinking of Proteins

Carbodiimides are zero length crosslinking agents which mediate amide bond formation between carboxylie aeid groups and amino groups present in the protein. The crosslinking reaction can be intramolecular or intermolecular. Intramolecular crosslinking is often used 

To reduee intramolecular coupling to lysine residues, the carbodiimide mediated coupling should be performed in a concentrated protein solution at a low pH using a large excess of the nueleophile. 

Chemical crosslinking of proteins in combination with mass spectroscopy is often used for the mapping of the three dimensional structures in proteins. An example is the crosslinking of hemoglobin. The authors also studied the structures of crosslinked neurofibrillary tangles isolated from the brain of an Alzheimer s disease patient. 

Monobifunctional crossUnkers, synthesized in the carbodiimide mediated esterification of dicarboxylic acids in the presence of sodium N-hydroxysuccinimide-3-sulfonate, are used in the mapping of protein structures.  

The treatment of collagen fibers with carbodiimides leads to an increase in viscosity. Dendrimers, such as polypropyleneimine octaamine, are used in combination with EDC to crosslink collagen.  

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Open-chain ligands were the first evaluated for complexation studies with indium and yttrium. The use of diethylenetriaminepentaacetic acid (DTPA) anhydride permitted early evaluation of labeled chelate-conjugates (Figure 2).80 The use of this activated chelating agent was quite popular, until the drawbacks associated with its crosslinking of proteins became apparent. 

The following protocol is designed for treating cells with the PIR reagent to study protein interactions in vivo. It is based on the method of Tang et al. (2005). The use of the PIR compound to treat intact cells results in the crosslinking of proteins both on the cell surface and within the cell, which indicates that the reagent is able to cross the cell membrane. 

Ru(ll)bpy32+ for Light-Triggered, Zero-Length Crosslinking of Protein Interactions... 

Pepinsky, R.B., Cappiello, D., Wilkowski, C., and Vogt, V.M. (1980) Chemical crosslinking of proteins in avian sarcoma and leukemia viruses. Virology 102, 205-210. 

Pyrrolidation and crosslinking of proteins can be considered biomarkers of either exposure or effect and are discussed in the previous section. 

The noncovalent bonding Involved In the crosslinking of protein gels Include 1) hydrogen bonding at specific sites, 2)... 

The efficient browning of proteins by 3-DG and MGO under anaerobic conditions emphasises that oxidation is not essential for Maillard reactions in vivo, although oxygen and oxidation appear to be rate-limiting for browning and crosslinking of proteins by aldo- and ketohexoses, as well as ascorbate. 

J. E. Litchfield, S. R. Thorpe, and J. W. Baynes, Oxygen is not required for the browning and crosslinking of protein by pentoses Relevance to Maillard reactions in vivo, Int. J. Biochem. Cell Biol, 1999, 31, 1297-1305. 

Lorenzen, P.C., Mautner, A., and Schlimme, E. 2000. Enzymatic crosslinking of proteins in the production of milk products In Proceedings of the 1st International Symposium on Enzymatic Protein Processing (H. Gruppen and W. van Hartingsveldt eds), pp. 163—169. TNO Nutrition and Food Research Institute, Zeist, The Netherlands. 

We now well appreciate, of course, that polymers are virtually everywhere. Some of them occur naturally, and we continue to better understand their compositions, structures, and properties. Many of these materials have been used since the dawn of human existence, for food, obviously. Cellulose alone has been essential for clothing, fire, shelter, tools, weapons, writing, and art. Leather is probably the result of the first synthetic polymer reaction, essentially the crosslinking of protein (elastin). How we progressed over time to the Polymer Age is a fascinating series of stories, some of which are well worth recounting here. 

Feeney and Whitaker (30, 31) have emphasized the importance of the enzymes transglutaminase, lipoxygenase, polyphenol oxidase and peroxidase in the crosslinking of proteins. With transglutaminase, chimeric proteins can be made readily by crosslinking two proteins, each with quite different properties (32, 33). 

Carbodiimides are often used in the crosslinking of proteins. They are a family of zero length protein crosslinking agents, which promote the formation of covalent crosslinks between reactive side groups of amino acids, but do not remain as part of the crosslinks. 

At the age of genomics and proteomics, carbodiimide mediated protein engineering is of considerable importance. Natural proteins and enzymes are readily modified or crosslinked to influence their behavior. The carbodiimide mediated inter- or intramolecular crosslinking of proteins is highly useful because no foreign molecules are introduced. Carbodiimides are zero length crosslinkers. Peptide based pharmaceuticals use carbodiimides in their manufacture and this market will increase considerably over the current low level. 

Further examples of Mannich reactions aimed at the modification of materials range from the use of suitable aldehyde mixtures in the crosslinking of proteins for contact Icnscs-- to the modification of rcsols with arylamines - for obtaining rapidly curing resins employed as wood adhesives. Some further examples of the functionalization of macromolecular derivatives for modifying the final properties of the material are found in Chap. Ill, D. 

Byrne, M.P., Broomfield, C.A., Stites, W.E. (1996). Mustard gas crosslinking of proteins through preferential alkylation of cysteines. J. Protein Chem. 15 131-6. 

Crosslinking of protein monolayers by mercuric ion (MacRitchie, 1970) and silicic acid (Minones et al., 1973) has been reported. These studies are relevant to poisoning by heavy-metal ions and to silicosis, effects that seem likely to result from attack on the cell membrane proteins. Crosslinking by mercuric ion was detected by a spectacular increase in surface viscosity and a decrease in compressibility when a number of proteins (BSA, insulin, ovalbumin, and hemoglobin) were spread on 0.001 M mercuric chloride solution. Poly-DL-alanine was unaffected whereas poly-L-lysine and poly-L-glutamic acid were affected in a similar manner to the proteins, indicating that mercuric ion interacts with the ionizable carboxyl and amino groups on the protein side—chains. Silicic acid similarly caused protein monolayers... 

Valentine et al. (1993) have shown that the initial dithiocarbamate protein adduct decomposes to isothiocyanate derivatives which then react with protein nucleophiles resulting in crosslinking. The crosslinking of protein in the nerve axons to cause their ultimate degeneration is correlated with the crosslinking of spectrin, a blood cell membrane protein. This suggests that the latter can be used as a biomarker of adverse effect of nerve damage. 

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