Fluorometric quantitation of cellular and nonprotein thiols.
A microfluorometric assay for thiols has been developed using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM). The technique may be used to quantitate either cellular or plasma thiols over a range of 0.01 to 3.0 nmol and may be used with as few as 1-3 X 10(5) cells giving highly proportional and reproducible results. Values for nonprotein thiols obtained with this assay agree well with previous reports on glutathione (GSH) levels for both lymphocytes and plasma. Readings are determined with the aid of an automated fluorescence microplate reader which allows up to 96 samples, including standards, to be read at the same time. Cellular thiols accessible after lysis were also quantitated before and after treatment of intact cells with various thiol-reactive chemicals. Interestingly, HgCl2, bromoethanesulfonic acid, and N-ethylmaleimide differentially modified protein and nonprotein thiol levels.
F C
FC
Ayers
G L
GL
Warner
K L
KL
Smith
D A
DA
Lawrence
D000818:Animals; D003374:Coumarins; D005033:Ethylmaleimide; D005260:Female; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D005978:Glutathione / Q000032:analysis / Q000097:blood; D006801:Humans; D051379:Mice; D008808:Mice, Inbred CBA; D011506:Proteins / Q000032:analysis; D013154:Spleen / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000097:blood
D003374:Coumarins; D005456:Fluorescent Dyes; D011506:Proteins; D013438:Sulfhydryl Compounds; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D005978:Glutathione; D005033:Ethylmaleimide