Sulfhydryl residue

Stop the conjugation reaction by capping residual sulfhydryls with the addition of NEM at a final concentration of 50 pAf Dissolve 6 mg of NEM in 1 mL DMSO and dilute 1 1000 in the conjugate reaction mixture. Incubate for 30 min at room temperature or overnight at 4°C (see Note 8) The conjugate is now ready for final purification... 

With the development of the amperometric and polarographic techniques, there have been made available methods for the determination of —SH groups of great actual and potential usefulness. The amperometric methods depend primarily on diver mercaptide formation. The polarographic methods may be applied either directly to the estimation of —SH groups or indirectly to the estimation of residual sulfhydryl-characteridng reagents added in excess. 

For CNBE, modification of the purified CNBE by DEPC shows that 1 mole of histidine residue existed in the active site of the enzyme. Modification by NBS shows that tryptophan residues existed in the active site and there is at least 1 mole of tryptophan residue in the substrate binding site. Results of modification by Ch-T and EDAC indicate that methionine residues and carboxyl groups were essential groups of the purified enzyme. In addition, hydroxyl groups, arginine residues, tyrosine residues, sulfhydryl groups, and disulfide bonds were not essential groups of the purified enzyme [100],... 

A comparison of the structures of penicillin and Dalanyl-Dalanine (cf. structures 41 and 42) shows that there is a great deal of similarity between the two molecules. Penicillin is essentially an acylated cyclic dipeptide of Dcysteine and Dvaline (84). As such, it contains a peptide bond, that of the /3-lactam ring, that can acylate the enzyme. Labeling studies of the peptidoglycan transpeptidase of Bacillus subtilis indicate that radioactive penicillin reacts with a sulfhydryl group of a cysteine residue of the enzyme (86). 

Some proteins contain covalent disulfide (S— S) bonds that link the sulfhydryl groups of cysteinyl residues. Formation of disulfide bonds involves oxidation of the cysteinyl sulfhydryl groups and requires oxygen. Intrapolypeptide disulfide bonds further enhance the stability of the folded conformation of a peptide, while interpolypeptide disulfide bonds stabilize the quaternary structure of certain oligomeric proteins. 

There are four disulfide bonds in short-chain (Type I) neurotoxins. This means that there are eight half-cystines. However, all Hydrophiinae toxins have nine halfcystines with one cysteine residue. An extra cysteine residue can be readily detected from the Raman spectrum as the sulfhydryl group shows a distinct S-H stretching vibration at 2578 cm" Some Laticaudinae toxins do not have a free cysteine residue as in the cases of L. laticaudata and L. semifasciata toxins. In long toxins (Type II) there are five disulfide bonds (Table III). 

The amino acid residues in neurotoxins which are important for neurotoxic action are still not entirely clarified. Some neurotoxins contain one free SH group, while others do not. From this fact, it would be logical to assume the sulfhydryl group is not essential. This was actually proven to be the case. 

Indirect evidence for the existence of different conformations of H,K-ATPase has been gained by site-selective reagents. Eor example, Schrijen et al. [67,95] demonstrated that Mg increased exposure of an essential arginine residue near the ATP-binding site and Mg " caused an increase in the number of reactive sulfhydryl groups on the enzyme. 

This same type of modification strategy also can be used to create highly reactive groups from functionalities of rather low reactivity. For instance, carbohydrate chains on glycoproteins can be modified with sodium periodate to transform their rather unreactive hydroxyl groups into highly reactive aldehydes. Similarly, cystine or disulfide residues in proteins can be selectively reduced to form active sulfhydryls, or 5 -phosphate groups of DNA can be transformed to yield modifiable amines. 

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