Linking reagents

In most cases, the proteia is immobilized onto y-aminopropyl sUica and covalently attached usiag a cross-linking reagent such as -carbonyl diimidazole. The tertiary stmcture or three dimensional organization of proteias are thought to be important for their activity and chiral recognition. Therefore, mobile phase conditions that cause proteia "deaaturatioa" or loss of tertiary stmcture must be avoided. 

AH of the reactions considered to be useful in the production of hemoglobin-based blood substitutes use chemical modification at one or more of the sites discussed above. Table 2 Hsts the different types of hemoglobin modifications with examples of the most common reactions for each. Differences in the reactions are determined by the dimensions and reactivity of the cross-linking reagents. Because the function of hemoglobin in binding and releasing... 

Two chemical approaches that involve nucleophilic substitution of the chlorine atoms attached to boron with linking reagents are reported in the literature. In both methods, the driving force is the formation of a stable by-product or one that can easily be stabilized. These substances contain chlorine. They can be distinguished by the number of steps, either one or two, required for preparing the polymer, starting from /i-chloroborazine. 

These one-step reactions display the following characteristics (1) the nitrogen-containing linking reagent is reactive at a relatively low temperature, (2) the reagents should be mixed carefully with the R-chloroborazine in such a way as to prevent the... 

Two-step synthetic routes to poly(/i-aminoborazines) from /i-chloroborazines involve initial nucleophilic reaction of the /i-chloroborazine with appropriate linking reagents followed by a deamination reaction of the as-obtained /i-aminoborazine. The 5-tiichloroborazine undergoes nucleophilic attack by ammonia or amine derivatives on the boron atom linked to chlorine atoms. For the same reasons previously quoted a tertiary amine (e.g., Et3N) must be added to precipitate the corresponding hydrochloride. 

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. 

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. 

Bloxham, D.P., and Sharma, R.P. (1979) The development of 5,5 -polymethylenebis(methanethiosulfona tes) as reversible cross-linking reagent for thiol groups and their use to form stable catalytically active cross-linked dimers with glyceraldehyde-3-phosphate dehydrogenase. Biochem. J. 181, 355. 

Brinkley, M. (1992) A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjugate Ghem. 3, 2. 

Chamow, S.M., Kogan, T.P., Peers, D.H., Hastings, R.C., Byrn, R.A., and Ashkenazi, A. (1992) Conjugation of soluble CD4 without loss of biological activity via a novel carbohydrate-directed cross-linking reagent. Biol. Ghem. 267, 15916-15922. 

Collioud, A., Clemence, J.-F., Sanger, M., and Sigrist, H. (1993) Oriented and covalent immobilization of target molecules to solid supports Synthesis and application of a light-activatable and thiol-reactive cross-linking reagent. Bioconjugate Chem. 4, 528-536. 

Davies, C.E., and Kaplan, J.G. (1972) Use of diimidoester cross-linking reagent to examine the subunit structure of rabbit muscle pyruvate kinase. Can. J. Biochem. 50, 416-422. 

Davies, C.E., and Stark, G.R. (1970) Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc. Natl. Acad. Sci. USA 66, 651. 

Denney, J.B., and Blobel, G. (1984) 1251-Labeled cross-linking reagent that is hydrophilic, photoactivat-able, and cleavable through an azo linkage. Proc. Natl. Acad. Sci. USA 81, 5286-5290. 

Jaffe, C.L., Lis, H., and Sharon, N. (1980) New cleavable photoreactive heterobifunctional cross-linking reagents for studying membrane organization. Biochemistry 19, 4423. 

Lee, W.T., and Conrad, D.H. (1985) The murine lymphocyte receptor for IgE. III. Use of chemical cross-linking reagents to further characterize the B lymphocyte Fee receptor. J. Immunol. 134, 518-525. 

Lewis, R.V., Roberts, M.F., Dennis, E.A., and Allison, W.S. (1977) Photoactivated heterobifunctional cross-linking reagents which demonstrate the aggregation state of phospholipase A2. Biochemistry 16, 5650-5654. 

Lomant, A.J., and Fairbanks, G. (1976) Chemical probes of extended biological structures synthesis and properties of the cleavable cross-linking reagent [35S] dithiobis(succinimidyl propionate)./. Mol. Biol. 104, 243-261. 

Luduena, R.F., Roach, M.C., Trcka, P.P., and Weintraub, S. (1982) Bioiodoacetyldithioethylamine A reversible cross-linking reagent for protein sulfhydryl group. Anal. Biochem. 117, 76. 

Mullet D.R. et al. (2001) Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis. Anal. Chem. 73, 1927-1934. 

Murayama, Y., Satoh, S., Oka, T., Imanishi, J., and Noishiki, Y. (1988) Reduction of the antigenicity and immunogenicity of xenografts by a new cross-linking reagent. ASAIO Trans. 34, 546. 

Peters, K., and Richards, F.M. (1977) Chemical cross-linking reagents and problems in studies of membrane structure. Annu. Rev. Biochem. 46, 523-551. 

Shephard, E.G., DeBeer, F.C., von Holt, C., and Hapgood, J.P. (1988) The use of sulfosuccinimidyl-2-(p-azidosalicylamido)-l,3 -dithiopropionate as a cross-linking reagent to identify cell surface receptors. Anal. Biochem. 168, 306-313. 

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

Staros, J.V. (1988) Membrane-impermeant cross-linking reagents Probes of the structure and dynamics of membrane proteins. Acc. Chem. Res. 21, 435-441. 

Sun, T.T., Bollen, A., Kahan, L., and Traut, R.R. (1974) Topography of ribosomal proteins of the Escherichia coli 30S subunit as studied with the reversible cross-linking reagent methyl 4-mercaptobu-tyrimidate. Biochemistry 13, 2334—2340. 

Wiley, D.C., Skehel, J.J., and Waterfield, M. (1977) Evidence from studies with a cross-linking reagent that the haemagglutinin of influenza virus is a trimer. Virology 79, 446-448. 

Willingham, G.L., and Gaffney, B.J. (1983) Reactions of spin-label cross-linking reagents with red blood cell proteins. Biochemistry 22, 892. 

Yan, M., Cai, S.X., Wybourne, M.N., and Keana, J.F.W. (1994) N-Hydroxysuccinimide ester functionalized perfluorophenyl azides as novel photoactivatable heterobifunctional cross-linking reagents. The covalent immobilization of biomolecules to polymer surfaces. Bioconjugate Chem. 5, 151-157. 

Zara, J.J. et al. (1991) A carbohydrate-directed heterobifunctional cross-linking reagent for the synthesis of immunoconjugates. Anal. Biochem. 194, 156-162. 

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