Disulfide cross-linking

Originally, vulcanization implied heating natural rubber with sulfur, but the term is now also employed for curing polymers. When sulfur is employed, sulfide and disulfide cross-links form between polymer chains. This provides sufficient rigidity to prevent plastic flow. Plastic flow is a process in which coiled polymers slip past each other under an external deforming force when the force is released, the polymer chains do not completely return to their original positions. 

Hydrogen bonding stabilizes some protein molecules in helical forms, and disulfide cross-links stabilize some protein molecules in globular forms. We shall consider helical structures in Sec. 1.11 and shall learn more about ellipsoidal globular proteins in the chapters concerned with the solution properties of polymers, especially Chap. 9. Both secondary and tertiary levels of structure are also influenced by the distribution of polar and nonpolar amino acid molecules relative to the aqueous environment of the protein molecules. Nonpolar amino acids are designated in Table 1.3. 

Keratin is the protein of hair and wool. These proteins are insoluble because of the disulfide cross-linking between cystine units. Permanent waving of... 

Sulfur-containing chemicals such as dimorpholinyl disulfide (DTDM) and tetraethylthiuram disulfide (TMTD) are not only effective accelerators, but they can also be used as sulfur donors. As such, they are effective ia controlling sulfur cross-link length to form primarily moao- and disulfide cross-links. These short cross-links are more thermally stable than conventional sulfur curing and thereby provide better heat and set resistance. 

Eig. 8. Sulfiir-based cure system designs where conventional systems are polysulfidic, EV systems are mono-/disulfidic, and semi-EV systems are clean polysulfidic. A shows pendent sulfide groups terminated by accelerator B, monosulftde cross-links C, disulfide cross-links D, polysulftde cross-links... 

In contrast, the EV cure systems employ much lower levels of free sulfur (0.1—1.0 phr) or they use sulfur donors such as TMTD or DTDM combkied with higher accelerator levels. The short mono- and disulfide cross-links that form often do not exhibit the excellent physical properties of the conventional systems but they do retain thek properties much better after aging. 

Fillers. Materials used as fillers (qv) in mbber can also be classified as acidic, basic, or neutral. Furnace blacks, ie, HAF, FEF, or SRF, are somewhat basic. As such, they can have an activating effect on sulfur cure rates. Furthermore, carbon blacks have been found to promote formation of mono/disulfide cross-links thereby helping minimize reversion and enhance aging properties. 

A sulfur donor system in NR (DTDM, 1.5 CBS, 1.5 TMTD, 0.5) yields a stable network stmcture with a contribution of 80% mono- and disulfidic cross-links at the optimum cure at 143°C or 183°C curing [13]. 

Cross-links, which impose strong conformational constraints on the intervening segment of the chain, generally are not classified as elements of secondary or tertiary structure. Disulfide cross-links in protein may certainly stabilize both secondary and tertiary structure, and such cross-links have the... 

Figure 4 Examples of long-range interactions in protein a disulfide cross-link and a polypeptide turn.

Tanford (1968) reviewed early studies of protein denaturation and concluded that high concentrations of Gdm-HCl and, in some cases, urea are capable of unfolding proteins that lack disulfide cross-links to random coils. This conclusion was largely based on intrinsic viscosity data, but optical rotation and optical rotatory dispersion (ORD) [reviewed by Urnes and Doty (1961) ] were also cited as providing supporting evidence. By these same lines of evidence, heat- and acid-unfolded proteins were held to be less completely unfolded, with some residual secondary and tertiary structure. As noted in Section II, a polypeptide chain can behave hydrodynamically as random coil and yet possess local order. Similarly, the optical rotation and ORD criteria used for a random coil by Tanford and others are not capable of excluding local order in largely unfolded polypeptides and proteins. The ability to measure the ORD, and especially the CD spectra, of unfolded polypeptides and proteins in the far UV provides much more incisive information about the conformation of proteins, folded and unfolded. The CD spectra of many unfolded proteins have been reported, but there have been few systematic studies. 

Fig. 4.12(a). An outline structure of a protein (here the enzyme phospholipase A2), showing a-helical runs of amino acids as cylinders (A-E) and anti-parallel P-sheet runs as heavy black arrows. Disulfide cross-links are shown (the enzyme is extracellular), and runs of no a/p secondary structure appear as thin lines. The structure is relatively immobile, and binds calcium in a constrained loop. (Reproduced with permission from Professor J. Drenth.)... 

Hamdan, F. F., Ward, S. D., Siddiqui, N. A., Bloodworth, L. M., and Wess, J. (2002) Use of an in situ disulfide cross-linking strategy to map proximities between amino acid residues... 

Shivaprasad, S., and Wetzel, R. (2004). An intersheet packing interaction in A beta fibrils mapped by disulfide cross-linking. Biochemistry 43, 15310-15317. 

S., Lerner-Marmarosh, N., Rousseau, M.E., Gros, P. and Senior, A.E. (2001) Cysteines 431 and 1074 are responsible for inhibitory disulfide cross-linking between the two nucleotide-binding sites in human P-glycoprotein. Journal of Biological Chemistry, 276, 26980-26987. 

Dow, L.K., Jones, D.N., Wolfe, S.A., Verdine, G.L., and Churchill, M.E.A. (2000) Structural studies of the high mobility group globular domain and basic tail of HMG-D bound to disulfide cross-linked DNA. Biochemistry 39, 9725-9736. 

Usually a mono- or disulfide cross-link occurs but larger numbers of sulfur atoms are possible. If the total percentage of sulfur in the material is <5%, it is usually very elastic. If >5% of sulfur is added, it produces a very hard, dark, nonelastic material called ebonite, sometimes used for things like combs and buttons. 

The 1 1 mixtures of strands 19 and 20 (0.5 mM each) were prepared in CH2CI2, methanol, and water. The samples were subjected to redox conditions (I2) for various lengths of time and then examined by matrix assisted laser desorption ionization (MALDI). As expected, 19 and 20 sequence specifically associated with each other in CH2CI2, leading to the disulfide cross-linked 19-20 as the major product. With an increasing ratio of methanol in CH2CI2, the cross-linked 19-20 stUl appeared as the dominant product. The same phenomenon was observed in pure methanol and even in water (Fig. 9.14b). The sequence dependence of the cross-linking of 19 and... 

Figure 9.17 Two components bearing complementary H bonding sequences can reversibly form disulfide cross-linked products. A-B represents the most stable product because of the stabilization fi om its fully matched, interstrand (now intramolecular) H bonds.

Figure 9.18 Partial 2-D H-NMR spectra of disulfide cross-linked 26-27 in (a) CDCI3 (rotational nuclear Overhauser effect spectroscopy, 278 000, 0.3-s mixing time) and (b) H20/THF-(7g (80/20, v/v NOESY, 278 000, 0.3-s mixing time). The same interstrand NOEs were detected in both CDCI3 and H20/THF-ii8.

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