Disulfide bridges cleavage

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins, (a) Oxidative cleavage by reaction with performic acid, (b) Reductive cleavage with snlfliydryl compounds. Disulfide bridges can be broken by reduction of the S—S link with snlfliydryl agents such as 2-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced —SH groups to re-establish disulfide bonds is a likelihood, S—S reduction must be followed by —SH modification (1) alkylation with iodoac-etate (ICH,COOH) or (2) modification with 3-bromopropylamine (Br— (CH,)3—NH,). 

Figure 51-3. Diagrammatic representation (notto scale) of prothrombin. The amino terminal is to the left region 1 contains all ten Gla residues. The sites of cleavage by factor Xa are shown and the products named. The site of the catalytically active serine residue is indicated by the solid triangle. The A and B chains of active thrombin (shaded) are held together by the disulfide bridge.

Another crosslinker, SAED (Chapter 5, Section 3.9), can be used in a similar fashion, but instead of transferring a radioactive label, it contains a fluorescent portion that is transferred to a binding molecule after cleavage. Similarly, sulfo-SBED routinely is used to study protein interaction. Cleavage of a disulfide bridge after capture of interacting proteins results in transfer of a biotin label to the unknown prey protein (Chapter 28, Section 3.1). The biotin modification then can be used to detect or isolate the unknown interactor for subsequent identification. 

Using a similar approach, Clq has been modified with biotin-HPDP and allowed to interact with its specific receptor. Subsequent purification of the Clq receptor was accomplished through cleavage of the disulfide bridge of the biotinylation reagent (Ghebrehiwet et al., 1988). 

It can be assumed that the amino acids following this hinge region (Val 93 to Leu 447) are part of the head domain. The point of papain cleavage is at amino acid 82 27. TTie core part of the polypeptide chain is mainly folded in )3-sheets (34 %) and to a lesser extent (15 %) arranged in alpha-helical structures 7. In contrast with CBH I the core of CBH II possesses only 2 disulfide bridges (176-235 368-415) and four free sulfhydryl groups. Similarly to CBH I carboxyl functions are involved in the active center (Asp 175 and Glu 184) 28. 

Fia. 2. Fractionation of human serum high-density lipoprotein ap>oprotein (apo HDD, scheme 2. Such a procedure takes advantage of the dimer— monomer conversion of fraction IV induced by the cleavage of the single disulfide bridge. R-IV = reduced fraction IV 8ME — 3-meroaptoethanol. Peaks — fraction HI = fraction IV fraction V. 

Mix samples with one-third of their volume of buffer G and heat at about 70 °C for 5 min. For cleavage of disulfide bridges add DTT to a final concentration of 10 mM. 

Cleavage of the Trt group of one chain 54 with a weak acid to give 55 and its subsequent thiolysis of the. S -SPy derivative of the second chain 57 directs the formation of the first interchain disulfide bond in 58. The second interchain disulfide bridge is formed between the two Acm-protected cysteine residues of the [bis(Acm), bis(tBu), mono-disulfide]-hetero-dimer 58 by treatment with iodine. Finally, treatment of 59 with chlorosilane/sulfoxide produces the third disulfide bond between the two tBu-protected cysteine residues yielding human insulin (42). 

Among the methods established in cysteine chemistry for cleavage of the Mob group with concomitant formation of disulfide bridges, i.e. I2, Tl( III )trifluoroacetate140 and DMSO/ TFA,[411 only the DMSO/TFA procedure allows for clean deprotection of SeC(Mob) residues and for their concomitant oxidation to selenocystine dimers or intrachain cyclic structures. IP°33]... 

As the ribosome synthesizes a new peptide chain, the chain usually begins to fold, creating regions of secondary and even incomplete tertiary structure. Enzymes then act on these folded residues to modify them. As noted above, an important modification that occurs at this stage is the formation of disulfide bridges. Another is the cleavage of the peptide backbone at specific sites, which may be important for the transport of the protein across membranes in the cell. These modifications occur during the process of translation, and so they are described as cotranslational modifications. 

Cleavage of Disulfide Bridges 147 5. Other Information from NMR Spectra... 

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