Cross-linking, protein

The key to hexavalent chromium s mutagenicity and possible carcinogenicity is the abiHty of this oxidation state to penetrate the cell membrane. The Cr(VI) Species promotes DNA strand breaks and initiates DNA—DNA and DNA-protein cross-links both in cell cultures and in vivo (105,112,128—130). The mechanism of this genotoxic interaction may be the intercellular reduction of Cr(VI) in close proximity to the nuclear membrane. When in vitro reductions of hexavalent chromium are performed by glutathione, the formation of Cr(V) and glutathione thiyl radicals are observed, and these are beHeved to be responsible for the formation of the DNA cross-links (112). 

Cross-linkage - bifunctional agents may form covalent bonds with each of two adjacent guanine residues and such inter-strand cross-links will lead to inhibition of DNA replication and transcription. Intra-strand and DNA-protein cross-links may also be formed ... 

Chromosomal aberrations Gene mutation Dominant lethal mutation Micronucleus formation Micronucleus formation Micronucleus formation Chromosomal aberrations Sister chromatid exchange Micronucleus formation Chromosomal aberrations Sister chromatid exchange DNA-protein cross-links Nondisjunction of Y chromosome in sperm DNA damage (single-strand breaks)... 

Brownlee, M., Vlassara, H., Kooney, A., Ulrich, P. and Cerami, A. (1986). Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking. Science 232, 1629-1632. 

Nackerdien, Z., Rao, G., Cacciuttolo, M.A., Gajewski, E. and Dizdaroglu, M. (1991). Chemical nature of DNA-protein cross-links produced in mammalian chromatin by hydrogen peroxide in the presence of iron or copper ions. Biochemistry 30, 4872-4879. 

Interestingly, the nucleophilic addition of water in the sequence of events giving rise to 41 represents a relevant model system for investigating the mechanism of the generation of DNA-protein cross-links under radical-mediated oxidative conditions [80, 81]. Thus, it was shown that lysine tethered to dGuo via the 5 -hydroxyl group is able to participate in an intramolecular cyclization reaction with the purine base at C-8, subsequent to one electron oxidation [81]. 

NA isolation and molecular characterization will be important to define the origin and functions of these proteins. At this time, infected cell nuclei offer the only source of these proteins, and NA have proved resistant to classic nuclear extraction methods (Yao and Jasmer, 1998). NA can be solubilized under conditions that co-extract nuclear lamins a/c and b (4 M urea, pH 8.0). Despite these similar physical properties, NA do not co-localize with lamins in the nucleoskeleton. However, both disulphide bonds and ionic interactions appear to contribute to nuclear complexes containing NA. In addition, NA can be cross-linked within host nuclei with protein cross-linking reagents. The foregoing properties represent current information available for the development of strategies to isolate and characterize these proteins and to investigate host proteins with which NA interact. 

Although formaldehyde penetrates very quickly, its protein cross-linking fixative effects are not as immediate. Modifications to this and improvements on the model used originally in studies by Baker et al.,7 Fox et al.,8 and Helander9 have demonstrated that in tissue and in these more complex models, the penetration and therefore Medawar s constant for formaldehyde is not as great as 5.5 and is probably more like 3.6.7... 

Finally temperature and tissue type do have an effect on the penetration and fixation. This, combined with a failure to appreciate protein cross-linking time or actual fixation time required for formaldehyde, is probably the main reason for intra- and interlaboratory variation. 

A second type of protein cross-link has been proposed to occur between a secondary amine and a carbonyl compound through the Mannich reaction20... 

These thermal analysis studies serve to establish a direct relationship between a heat-induced AR method and the reversal of formalin-induced intra- and intermolecular protein cross-links.10 2831 Further, while formalin-treatment provides thermal stability to RNase A, this stabilization is not sufficient to prevent thermally induced protein denaturation at temperatures (>100°C) typically used in heat-induced AR methods.32 34 The implications of this finding for the mechanism of AR will be discussed further in Section 15.6. 

Fraenkel-Conrat H, Olcott HS. Reaction of formaldehyde with proteins cross-linking of amino groups with phenol, imidazole, or indole groups. I. Biol. Chem. 1948 174 827-843. 

Figure 16.5 Immunostained peptide arrays after various treatments of fixation, protein cross-linking, and antigen retrieval, as indicated at the top. Each row has a different peptide that is immunoreactive for the antibody denoted to the left. Column A represents the baseline condition, without any treatment whatsoever. Column B shows immunoreactivity of each peptide after overnight formalin fixation. Column C shows the immunoreactivity after first coating the array with an irrelevant protein (casein) followed by overnight formalin fixation. Column D illustrates the immunoreactivity of the peptides after the treatment of column C, and then antigen retrieval. Reproduced with permission from Reference 15, 2006 American Society for Clinical Pathology.

These data suggest that the loss of immunoreactivity after formalin fixation involves a protein cross-linking reaction. The first amino acid is at or near the antibody epitope. The second can be on the same protein or on another nearby protein. A variety of different chemical reactions with formaldehyde can be occurring. The common theme is that regardless of the details of the formaldehyde-induced cross-linking reaction, steric interference prevents antibodies from gaining access to the epitope. 

Several mechanisms have been proposed to explain HIAR the cleavage of protein-protein cross-links,3,6 the disruption of cross-links involving calcium ions,5 an increase in tissue permeability for antibodies, and the removal of trace amounts of paraffin in the tissues. However, recent studies have indicated that the fundamental mechanism of HIAR is based on the cleavage of protein-protein cross-links. 

Toews J, Rogalski JC, Clark TJ, et al. Mass spectrometric identification of formaldehyde-induced peptide modifications under in vivo protein cross-linking conditions. Anal. Chim. Acta. 2008 618 168-183. 

Owing to the fact that organic substances have to be separated from a complex sample, transferred to the mass spectrometer target and desorbed, it seems impossible so far to analyse cross-linked binding media from artwork (e.g. dried oil, aged proteins, cross-linked synthetic polymers). Application of classical methods of sample treatment for... 

Blattler, W.A., Kuenzi, B.S., Lambert, J.M., and Senter, P.D. (1985a) New heterobifunctional protein cross-linking reagent that forms an acid-labile link. Biochemistry 24, 1517-1524. 

Ewig, R.A.C., and Kohn, K.W. (1977) DNA - protein cross-linking and DNA interstrand cross-linking by haloethylnitrosoureas in L1210 cells. Cancer Res. 38, 3197. 

Hall, D.B., and Struhl, K. (2002) The VP16 activation domain interacts with multiple transcriptional components as determined by protein-protein cross-linking in vivo.]. Biol. Chem. 277, 46043-46050. 

Liu, S.C., Fairbanks, G., and Palek, J. (1977) Spontaneous reversible protein cross-linking in the human erythrocyte membrane. Temperature and pH dependence. Biochemistry 16, 4066. 

Mentzer Jr., W.C., Lewis, S., Pennathur-Das, R., Halpin, R., Cerrone, K.L., Lubin, B., and Kenyon, G.L. (1982) Formation of 5-carbomethoxyvaleramidine during hydrolysis of the protein cross-linking agent dimethyl adipimidate./. Brotein Chem. 1, 141-155. 

Pandurangi, R.S. et al. (1998) Chemistry of bifunctional photoprobes Part 4. Synthesis of the chromoge-nic, cleavable, water soluble and heterobifunctional (N-Methyl amino perfluoroaryl azide benzamido)-ethyl-l,3-dithiopropionyl sulfosuccinimide An efficient protein cross-linking agent. Bioorg. Chem. 26(4), 201-212. 

Politz, S.M., Noller, H.F., and McWhirter, P.D. (1981) Ribonucleic acid-protein cross-linking in Escherichia coli ribosomes (4-azidophenyl) glyoxal, a novel heterobifunctional reagent. Biochemistry 20, 372-378. 

Srinivasachar, K., and Neville Jr., D.M. (1989) New protein cross-linking reagents that are cleaved by mild acid. Biochemistry 28, 2501. 

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