TY - JOUR
T1 - Determination of δ-aminolevulinate dehydratase activity by a specific fluorometric coupled-enzyme assay
AU - Giampietro, Philip F.
AU - Desnick, Robert J.
N1 - Funding Information:
The authorsw ish to thank Dr. David F. Bishop for providing helpful suggestions and Ms. Linda Lugo and Ms. Mary Ann Dent for their expert clerical assistance. This research was supported in part by a Grant (1 RO 1 AM26824) from the National Institutes of Health and a Grant (l-535) from the March of Dimes Birth Defects Foundation. P.F.G. was the recipient of a NIH Predoctoral Fellowship in Human Genetics (I T32 HD07 105).
PY - 1983/5
Y1 - 1983/5
N2 - A coupled-enzyme assay for the specific and sensitive determination of δ-aminolevulinate dehydratase activity has been developed. The assay specifically measured picomole quantities of the product, porphobilinogen, by its enzymatic conversion to uroporphyrinogen I and the fluorometric detection of oxidized uroporphyrin I. The coupled-enzyme assay was linear with time and protein concentration and required less than 3 h for 20 individual determinations. Under the standard assay conditions, 10 to 100 pmol of uroporphyrin I was reliably measured, representing 0.085 to 0.850 nmol/h of δ-aminolevulinate dehydratase activity per assay. In addition, the fluorometric assay was more sensitive than either the standard or the semimicro colorimetric methods. The specificity, rapidity, and sensitivity of this new fluorometric method facilitates the reliable determination of low levels of aminolevulinate dehydratase activity in small amounts of crude tissue homogenates or in cultured cells.
AB - A coupled-enzyme assay for the specific and sensitive determination of δ-aminolevulinate dehydratase activity has been developed. The assay specifically measured picomole quantities of the product, porphobilinogen, by its enzymatic conversion to uroporphyrinogen I and the fluorometric detection of oxidized uroporphyrin I. The coupled-enzyme assay was linear with time and protein concentration and required less than 3 h for 20 individual determinations. Under the standard assay conditions, 10 to 100 pmol of uroporphyrin I was reliably measured, representing 0.085 to 0.850 nmol/h of δ-aminolevulinate dehydratase activity per assay. In addition, the fluorometric assay was more sensitive than either the standard or the semimicro colorimetric methods. The specificity, rapidity, and sensitivity of this new fluorometric method facilitates the reliable determination of low levels of aminolevulinate dehydratase activity in small amounts of crude tissue homogenates or in cultured cells.
UR - https://www.scopus.com/pages/publications/0020606344
U2 - 10.1016/0003-2697(83)90138-0
DO - 10.1016/0003-2697(83)90138-0
M3 - Article
C2 - 6577805
AN - SCOPUS:0020606344
SN - 0003-2697
VL - 131
SP - 83
EP - 92
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -