Cross-Linking with Bifunctional Reagents

Hartman, F.C., and Wold, F. (1966) Bifunctional reagents. Cross-linking of pancreatic ribonuclease with a diimido ester. J. Am. Chem. Soc. 88, 3890-3891. 

We have observed that such proteins as CaM and bovine serum albumin (BSA) can be developed at the air-water interface to form monolayer protein films. In previous works, the developed BSA monolayer was stabilized by cross-linking with a bifunctional reagent immediately after the preparation of protein monolayer. The BSA thin film thus prepared can be employed as a passive material, e.g., an ultrathin protein film for a matrix of enzyme-linked immunosorvent assays. 

Chemical Immobilization 4.2.1. CROSS-LINKING WITH A BIFUNCTIONAL REAGENT... 

Cross-linking with a bifunctional reagent. Examples of such reagents are ethyl chloroformate [7], glutaraldehyde [8] or ethyl maleic anhydride [9]. Column properties of supports prepared this way are generally low, and due to the presence of unreacted functional groups in the matrix non-specific adsorption can be high. 

Enzyme cross-linked with bifunctional reagents... 

Immobilization of enzymes by cross-linking with bifunctional reagents is carried out by different methods direct cross-linking of enzymes cross-linking of enzymes with inert proteins adsorption of enzymes on a water-insoluble carrier with subsequent processing using a bifunctional reagent. Albumin is... 

The commercially available ceUnlose is generally cross-linked with a bifunctional reagent snch as epichlorohydrin... 

Enzyme Crystals and Precipitates. Solid enzyme preparations can be cross-linked with a bifunctional reagent to yield cross-linked enzyme crystals (CLEG) (21), or cross-linked enzyme aggregates (CLEA) (22) that are insoluble in aqueous as well as nonaqueous media. This maintains their catalytic activity and increases their thermostability and resistance to proteases. The activity of the preparation remains high because of the high purity of the protein. CLECs are not commercially available anymore. CLEAs can be dried and suspended in any type of solvent (23). 

Bis-Pyndoxal Tetraphosphate. A second class of bifunctional reagents, described in 1988, involves two pyridoxal groups linked by phosphates of different lengths (89). As shown in Table 4, the yield of intramolecularly cross-linked hemoglobin increases dramatically with increasing length of the phosphate backbone. It is beheved that the site of reaction of (bis-PL) is between the amino-terminal amino group of one P-chain and the... 

It has been found that DTBP cross-linking substantially increased the salt stability of the complexes. The salt stabilization is reversed upon the addition of DTT, which cleaves the bifunctional reagent, indicating that it is not due to the conversion of the amines to amidines and is dependent upon the cross-linking. Similar results were achieved with other polycations, including poly(allylamine), and histone HI. 

Hillel, Z., and Wu, C.W. (1977) Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents. Biochemistry 16, 3334-3342. 

Sato, S., and Nakao, M. (1981) Cross-linking of intact erythrocyte membrane with a newly synthesized cleavable bifunctional reagent./. Biochem. (Tokyo) 90, 1177. 

Smith, R.J., Capaldi, R.A., Muchmone, D., and Dahlquist, F. (1978) Cross-linking of ubiquinone cytoch-nome c reductase (complex III) with periodate-cleavable bifunctional reagents. Biochemistry 17, 3719-3723. 

Enzyme membranes can be prepared by adsorbing the enzyme on the surface of a suitable native or synthetic membrane, or, in the case of membranes with large pores, by impregnating the whole membrane with enzyme. The resulting enzyme membrane can be stabilized by covalently cross-linking the adsorbed protein with a suitable bifunctional reagent (8 ). 

Bifunctional reagents, such as bis-imido esters, have been widely used because they react under mild condidons specifically with the amino groups of proteins. Bifunctional imido esters that introduce a disulfide bond in the cross-link are especially useful since these bonds can be readily cleaved by reduction, allowing individual proteins to be regenerated. The reagent most commonly used for ribosomal proteins is 2-iminothiolane (Traut et al, 1980). 

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