C. Lefevre et al., TEXAS RED-X AND RHODAMINE RED-X, NEW DERIVATIVES OF SULFORHODAMINE-101 AND LISSAMINE RHODAMINE-B WITH IMPROVED LABELING AND FLUORESCENCE PROPERTIES, Bioconjugate chemistry, 7(4), 1996, pp. 482-489
Texas Red sulfonyl chloride (TR-SC) and Lissamine rhodamine B sulfonyl
chloride (LRB-SC) are popular dyes often used to prepare red fluoresc
ent conjugates that are useful second labels in combination with fluor
escein. Unfortunately, being sulfonyl chloride derivatives, both are u
nstable to moisture during storage and prone to hydrolysis in the conj
ugation reaction. Their instability causes the percentage of reactive
dye to vary from lot to lot and requires use of low temperatures and a
relatively high pH to optimize conjugation efficiency. Succinimidyl e
sters of the aminohexanoic acid sulfonamides of both dyes have been pr
epared, which are designated Texas Red-X succinimidyl ester (TR-X-SE)
and Rhodamine Red-X succinimidyl ester, respectively. Their spectral p
roperties are similar to those of their sulfonyl chloride analogs; mor
eover, incorporation of the succinimidyl ester at the end of the aliph
atic chain spacer facilitates conjugation, decreases precipitation of
proteins during conjugation and storage, and usually increases the flu
orescence yield of the conjugate. Comparison of the rate of hydrolysis
of TR-SC with that of TR-X-SE shows that, while the former was comple
tely hydrolyzed within 5 min by exposure to water, TR-X-SE retains mos
t of its reactivity for more than an hour. The reactivity of both new
derivatives is high between pH 7.5 and 8.5, allowing conjugation of pr
oteins that do not tolerate the high pH required for reaction with sul
fonyl chlorides. In addition, Texas Red maleimides and haloacetamides
containing spacer groups were prepared for labeling sulfhydryl groups.
A Texas Red-X derivative of phalloidin has also been prepared, and it
s use for labeling F-actin has been characterized.