Modification thiolation

If cellular redox state, determined by the glutathione status of the heart, plays a role in the modulation of ion transporter activity in cardiac tissue, it is important to identify possible mechanisms by which these effects are mediated. Protein S-,thiolation is a process that was originally used to describe the formation of adducts of proteins with low molecular thiols such as glutathione (Miller etal., 1990). In view of the significant alterations of cardiac glutathione status (GSH and GSSG) and ion-transporter activity during oxidant stress, the process of S-thiolation may be responsible for modifications of protein structure and function. 

Miller, R.M., Sies, H., Park, E.-M. and Thomas, J.A. (1990). Phosphorylase and creatine kinase modification by thiol-disulphide exchange and by xanthine oxidase-initiated S-thiolation. Arch. Biochem. Biophys. 276, 355-363. 

The introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

Figure 1.59 Thiolation of an amine-containing compound with methyl 3-mercaptopropionimidate. The modification preserves the positive charge on the primary amine.

The following protocol represents a generalized method for protein thiolation using SATA. For comparison purposes, contrast the variation of this SATA modification method as outlined in Chapter 20, Section 1.1 for use in the preparation of antibody-enzyme conjugates. 

Dissolve the protein to be thiolated at a concentration of l-5mg/ml in 50 mM sodium phosphate, pH 7.5, containing 1-10 mM EDTA. Other non-amine containing buffers such as borate, HEPES, and bicarbonate also may be used as the reaction medium. The effective pH for the NHS ester modification reaction is in the range of 7.0-9.0, but environments closer to neutrality will limit the hydrolysis of the ester. 

Thiolation of peptides and other small molecules containing amines proceeds easily with N-acetyl homocysteine thiolactone. However, protein modification often results in much lower yields unless the reaction is done for extended periods at pH 10-11. 

To remove the silver mercaptide formed from the facilitated protein thiolation reaction, add an excess of thiourea to convert all the silver into a soluble Ag(thiourea)2 complex and free the sulfhydryl modifications. 

PDPH also may be used as a thiolation reagent to add sulfhydryl functional groups to carbohydrate molecules. The reagent can be used in this sense similar to the protocol described for AMBH (Chapter 1, Section 4.1). After modification of an oxidized polysaccharide with the hydrazide end of PDPH, the pyridyl group is removed by treatment with DTT, leaving the exposed sulfhydryl (Figure 5.15). 

Although amine-reactive protocols, such as SATA thiolation, result in nearly random attachment over the surface of the antibody structure, it has been shown that modification with up to 6 SATAs per antibody molecule typically results in no decrease in antigen binding activity (Duncan et al., 1983). Even higher ratios of SATA to antibody are possible with excellent retention of activity. 

Figure 21.5 SPDP can be used to modify both an antibody and a toxin molecule for conjugation purposes. In this case, the antibody is thiolated to contain a sulfhydryl group by modification with SPDP followed by reduction with DTT. A toxin molecule is then activated with SPDP and reacted with the thiolated antibody to effect the final conjugate through a disulfide bond.

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