Disuccinimidyl esters as bifunctional crosslinking reagents for proteins: assays with myosin.

Bifunctional crosslinking reagents have been used in studies of the spatial arrangement of muscle contractile proteins, such as myosin and actin, either in their soluble forms [ 1,2], or in synthetic filaments [3-51, or even in myofibrils [3]. The most commonly used reagents are the bisimidates, which are very reactive, but also quite unstable in aqueous solution; incomplete substitution and unexpected side reactions furthermore occur if the crosslinking reaction is at pH -8 [6,7]. A disuccinimidyl ester, the dithiobis (succinimidyl propionate), DSP, that does not have these drawbacks and contains, moreover, an easily cleavable disulfide bond, has been described [8]; unlike the bis-imidates, this reagent allowed the crosslinking of the two heads of a myosin molecule [ 21. Owing to the high chemical reactivity of DSP and its stability in water, we thought it interesting to synthesize a series of disuccinimidyl esters of various chain lengths (table 1). These include non-cleavable reagents (compounds I-IV), and also reagents with either a uic-glycol (compounds V, VI) or an ethylenic bond (compound VII); the crosslinks formed by these last compounds can in principle be cleaved,