Blocking Sulfhydryl Groups

Cadmium (soft, Cd2+) Renal toxicity Blocks sulfhydryl groups in enzymes and competes with zinc. Stimulates metallothionein synthesis and interferes with Cu(II) and Zn(II) metabolism. 

Arsenic As Impairment of growth and reproduction (animals) Poisonous Blocks sulfhydryl groups of enzymes... 

The reason that Hb doesn t block the action of NO is due to a unique interaction between Cys93/3 of Hb and NO recently described by Li Jia, Celia and Joseph Bonaventura, andjohnathan Stamler at Duke University. Nitric oxide reacts with the sulfhydryl group of Cys93/3, forming an S-nitroso derivative ... 

Dissolve the macromolecule containing sulfhydryl groups to be blocked in a buffer having a pH of 6.5-7.5. Sodium phosphate (0.01-0.1M) at pH 7.2 works well. Avoid... 

Figure 1.120 The reaction of NEM with sulfhydryl groups yields a thioether derivative, permanently blocking the thiol.

Thus, iodoacetamide has the highest reactivity toward cysteine sulfhydryl residues and may be directed specifically for —SH blocking. If iodoacetamide is present in limiting quantities (relative to the number of sulfhydryl groups present) and at slightly alkaline pH, cysteine modification will be the exclusive reaction. For additional information on a-haloacetate reactivities and a protocol for blocking, see Section 4.2 (this chapter). 

To remove the sulfenylthiosulfate blocking group, add a 300-fold excess of DTT over the amount of blocked sulfhydryls present. Alternatively, add DTT to obtain a 0.01-0.1M final concentration. Cysteine also may be utilized to regenerate some enzymes to full activity. 

Methyl methanethiosulfonate (MMTS) is a small reversible blocking agent for sulfhydryl groups (Thermo Fisher, Toronto Research). It reacts with free thiols to form a dithiomethane modification with release of sulfinic acid (Figure 1.122). The sulfinic acid component decomposes into volatile products, which don t affect the disulfide formed from the MMTS reaction Alkylthiosulfonates react rapidly with thiols under mild conditions at physiological pH. The MMTS compound is a liquid at 10.6 M concentration and is conveniently added to a reaction medium by pipette. Complete thiol modifications of available cysteine residues in proteins can... 

To block unreacted sulfhydryl groups, add iodoacetamide to the solution to a final concentration of 2 mM. 

Iodoacetate derivatives have been used for decades to block or crosslink sulfhydryl groups in proteins and other molecules (Chapter 1, Section 5.2). At mildly alkaline pH values (pH 8-8.5), iodoacetyl derivatives are almost entirely selective toward the cysteine —SH groups in proteins. Disulfide reduction or thiolation reagents can be used to create the required sulfhydryl groups on proteins containing no free sulfhydryls. 

Lee et al. (1978) investigated the possibility that sulfhydryl groups were required in the formation of the clostridial inhibitor in cured meats. Sulfhydryl groups of meat proteins were blocked by treatment with silver lactate, then the samples were cooked with sodium nitrite before inoculation with C. botuli num spores. Botulinal growth as measured by gas and toxin production was similar to controls without silver lactate treatment. They thus concluded that sulfhydryl groups were not required for the antibotulinal effects of nitrite in cured meats. 

In addition to the alkylation with iodoacetate (Eq. 3-24), sulfhydryl groups can react with N-ethylmale-imide (Eq. 3-39).281 This reaction blocks the SH groups irreversibly and has often been used in attempts to establish whether or not a thiol group plays a role in the functioning of a protein. Loss of function in the... 

The ricin and abnn A chains used to block high affinity binding sites on the column should be alkylated to remove the free sulfhydryl group that could interfere with conjugate purification... 

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