Phenomenological analysis of the kinetics of the production of interchain disulfide cross-links in two-chain, coiled-coil proteins by reaction with 5,5'-dithiobis(2-nitrobenzoate).
In parallel, registered, two-chain coiled-coil proteins such as tropomyosin, paramyosin, or myosin rods, a cysteine may appear at the same level on each chain. These may be cross-linked by reaction with 5,5'-dithiobis(2-nitrobenzoate) (NbS2). This technique is useful in several types of studies of such proteins. It is generally accepted that the NbS2, ordinarily at high molar concentration relative to protein, first reacts in a pseudo-first-order process with one cysteine, blocking it by formation of a mixed disulfide (NbS-S-protein); then, the second cysteine either reacts similarly, producing a doubly blocked site, or attacks the mixed disulfide in a sulfhydryl-disulfide interchange to produce an interchain cross-link. Here, the coupled differential equations for such a system are first set up and solved for a molecule with one such cross-linkable site on the assumption that the condition of one sulfhydryl (blocked or unblocked) does not alter the kinetics of its neighbor. Solutions are presented, giving the concentrations of all species as a function of time and of the rate constants for blocking and for cross-linking. It is also shown how nonreducing sodium dodecyl sulfate/polyacrylamide gel electrophoresis experiments on the products allow a determination of the ratio of the two rate constants. The theory is applied to illustrative data on tropomyosin and shown to fit well. Values of both rate constants emerge. A similar analysis is made for the case where two independent cross-linkable sites exist per molecule. The equations are applied to extent data on short subunit 2 of myosin rod.(ABSTRACT TRUNCATED AT 250 WORDS)
D004220:Disulfides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008954:Models, Biological; D009579:Nitrobenzoates / Q000494:pharmacology*; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*
D004220:Disulfides; D046911:Macromolecular Substances; D009579:Nitrobenzoates; D011506:Proteins; D004228:Dithionitrobenzoic Acid; D004229:Dithiothreitol