Identification of resilin in the leg of cockroach, Periplaneta americana: confirmation by a simple method using pH dependence of UV fluorescence

We have examined the tarsus (foot) and tibial segments of the cockroach leg to identify structures that contain the elastic protein resilin. The prese nce of resilin was tested using the conventional criteria of fluorescent em ission at 420 nm under UV illumination and histological staining of wholemo unt tissues by toluidine blue. We have also developed a simple method of co nfirming identification of of resilin through changes in its fluorescence t hat occur with alteration of pH of the surrounding medium. Using a commonly available excitation filter that only passes light at. >330 nm, we found t hat the emission was present at neutral pH but was eliminated at low pH. It then reversibly reappeared when medium of higher pH was restored. This eff ect is attributable to a known shift in the absorption maximum of amino aci ds of resilin that occurs in acidic media (from 330 to 285 nm). The accurac y of this method of identification was confirmed by examination of ligament s of the wing hinge, which has previously been shown to contain resilin in a number of insects. Using there techniques, we have identified resilin in association with ligaments at the tibio-tarsal joint and in the articulatio n between the fourth and fifth tarsal segments of the leg. The anatomical a rrangement of these ligaments suggests that they could aid in the generatio n of leg movements during walking by functioning as elastic antagonists to the actions of leg muscles. The method of identification we have devised co uld readily be applied to aid in the localization of resilin in other anima ls. (C) 2000 Elsevier Science Ltd. All rights reserved.