Mercaptoethanol hydrogen bonding

Proteins that possess a quaternary structure are composed of several separate polypeptide chains held together by noncovalent interactions. When such proteins are examined under dissociating conditions (e.g., 8 M urea to weaken hydrogen bonds and hydrophobic interaction, 1 m/lf mercaptoethanol to disrupt disulfide bonds), the molecular weight of the component polypeptide chains can be determined. By comparison with the native molecular weight, it is often possible to determine how many polypeptide chains are involved in the native structure. 

Ribonuclease, the enzyme that hydrolyzes ribonucleic acids (Chap. 7), contains four disulfide bonds that help to stabilize its conformation. In the presence of 6 M guanidine hydrochloride, to weaken hydrogen bonds and hydrophobic interactions, and 1 mM mercaptoethanol, to reduce the disulfide bonds, all enzymatic activity is lost, and there is no sign of residual secondary structure. On removing the guanidine hydrochloride by dialysis or gel filtration, enzymatic activity is restored, the native conformation is regained, and correct disulfide bonds are reformed. 

Experiments on crosslinking have been carried out in the author s laboratory (93). Solutions of isolated carp actomyosin or myosin in 0.6 M KC1 or suspensions in 0.05 M KC1 have been stored at -20°C. Samples were taken at intervals and homogenized with several different solutions. The solutions used were water (to test for nonspecific association forces), 0.6 M KC1 (to test for ionic bonds), 0.5 M e-mercaptoethanol (to test for disulfide bonds), 1.5 M urea (to test for hydrogen bonds), 8 M urea (to test for hydrogen bonds and nonpolar bonds), and 1 M K0H (to test for ionic bonds and others) (135,136). Combinations of these solvents were also tested. 

Shagidullin, R.R., Chernova, A.V, and Plyamovatyi, A.Kh., Intramolecular hydrogen bonds and the conformations of mercaptoethanol molecules, Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, 711-714 (1989). 

Furthermore, it has been demonstrated that these mannoproteins share covalent bonds with glucane (Moine-Ledoux and Dubourdieu, 1999). They remain in the cell walls treated simultaneously with sodium dodecyl sulfate (SDS) (which cuts the hydrogen bonds) and /i-mercaptoethanol (Figure 1.21), which do not affect osidic bonds. 

In other applications, such as molecular weight determination or analysis of the protein structures, denaturation of the proteins is required and SDS or guanidine should be added to the buffer solution to disrupt hydrogen bonds, electrostatic interactions, and/or hydrophobic interactions. However, many proteins also contain disulfide linkages, which can compromise the complete dissociation of multimeric proteins. To obtain a complete and uniform denaturation, sulfhydryl reagents, such as 2-mercaptoethanol, should be used in order to break the S-S bonds by reduction. 

One further aspect besides H-atom abstraction must, however, be considered in the reactions of thiyl radicals with PUFAs, namely, the possibility of concurrent RS additions to the double bonds. By analyzing a particular system in which the thiyl radical from mercaptoethanol had reacted with linolenic acid, Schoneich at came to the conclusion that the abstraction of bisallylic hydrogen and thiyl addition occur with comparable rates. The C-centered adduct radical formed in the addition reaction is, of course, prone for repair in the presence of thiols, i.e., will subsequently regenerate thiyl radicals via the back reaction of equilibrium 14. These, in turn, will re-enter into the abstraction / addition competition cycle and eventually all of the thiyl radicals will appear to have reacted via the abstraction route. Experimentally, overall efficiencies of up to 85 % have been measured and the difference to the limiting 100% efficiency may be accounted for by side and termination processes. But looking into the details, the situation is, in fact, even more complex. As shown by Schwinn at al., there is still another process which readily occurs within the RS -adducts to the double bonds, namely, cis-trans isomerization. Quantification of this is of imminent interest for the biological community. 

The effects of pH, ionic strength, reagent concentration ratios and deuterium substitution of the sulfur-bonded hydrogens on both the rates and rate laws for the methylene blue oxidations of mercaptoethanol and dithioerythritol have been determined. A free radical chain mechanism consistent with the observed kinetic behavior of the oxidation reactions is proposed. A key feature of the proposed mechanism is the formation of the sulfur-sulfur linkage of the disulfide in the reversible formation of a disulfide radical anion (RSSR) as a chain propagating step in the chain sequence. 

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