| Abstract
| - Texas Red sulfonyl chloride (TR-SC) and Lissamine rhodamine B sulfonylchloride (LRB-SC) arepopular dyes often used to prepare red fluorescent conjugates that areuseful second labels incombination with fluorescein. Unfortunately, being sulfonylchloride derivatives, both are unstableto moisture during storage and prone to hydrolysis in the conjugationreaction. Their instability causesthe percentage of reactive dye to vary from lot to lot and requires useof low temperatures and arelatively high pH to optimize conjugation efficiency.Succinimidyl esters of the aminohexanoic acidsulfonamides of both dyes have been prepared, which are designatedTexas Red-X succinimidyl ester(TR-X-SE) and Rhodamine Red-X succinimidyl ester, respectively.Their spectral properties are similarto those of their sulfonyl chloride analogs; moreover, incorporation ofthe succinimidyl ester at theend of the aliphatic chain spacer facilitates conjugation, decreasesprecipitation of proteins duringconjugation and storage, and usually increases the fluorescence yieldof the conjugate. Comparisonof the rate of hydrolysis of TR-SC with that of TR-X-SE shows that,while the former was completelyhydrolyzed within 5 min by exposure to water, TR-X-SE retains most ofits reactivity for more thanan hour. The reactivity of both new derivatives is high between pH7.5 and 8.5, allowing conjugationof proteins that do not tolerate the high pH required for reaction withsulfonyl chlorides. In addition,Texas Red maleimides and haloacetamides containing spacer groups wereprepared for labelingsulfhydryl groups. A Texas Red-X derivative of phalloidin has alsobeen prepared, and its use forlabeling F-actin has been characterized.
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