Preparation and high-performance liquid chromatography of 3'-phosphoadenosine-5'-phospho(/sup 35/S)sulfate with a predetermined specific activity
3'-Phosphoadenosine-5'-phospho(35S)sulfate (PAP/sup 35/S) was prepared by incubating ATP and carrier-free H/sup 2/(/sup 35/)SO/sub 4/ with a 100,000g supernatant fraction prepared from chick embryo chondrocytes. The product was partially purified by paper electrophoresis and mixed with unlabeled PAPS to give a solution of PAP/sup 35/S with a specific activity and a concentration approximating those required for the desired metabolic studies. The product was analyzed by high-performance liquid chromatography on an anion-exchange column to determine the proportion of the /sup 35/SO/sub 4/ cpm and A260 material found in the PAPS and other contaminating nucleotides. The PAP/sup 35/S was purified further by preparative high-performance liquid chromatography. The exact specific activity of the PAP/sup 35/S was then determined by using this PAP/sup 35/S preparation as the SO/sub 4/ donor in a sulfotransferase reaction using a microsomal preparation from the chick embryo chondrocytes as the enzyme and an /sup 3/H-labeled oligosaccharide as the SO/sub 4/ acceptor. The sulfated oligosaccharide was then isolated and the number of /sup 3/H and /sup 35/SO/sub 4/ counts per minute in this product were used to calculate the specific activity of the donor. The features of this generally useful approach for preparing PAP/sup 35/S of any desired specific activity and concentration are discussed.
- Research Organization:
- Univ. of Illinois, Urbana
- OSTI ID:
- 5980227
- Journal Information:
- Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 2; ISSN ANBCA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
400702* -- Radiochemistry & Nuclear Chemistry-- Properties of Radioactive Materials
ANIMAL CELLS
ANIMAL TISSUES
ANIMALS
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIRDS
BODY
CARTILAGE
CHEMICAL PREPARATION
CHICKENS
CHROMATOGRAPHY
CONNECTIVE TISSUE
DAYS LIVING RADIOISOTOPES
ELECTROPHORESIS
EMBRYOS
EVEN-ODD NUCLEI
FOWL
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
NUCLEI
NUCLEOTIDES
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
RADIOISOTOPES
SEPARATION PROCESSES
SULFATES
SULFUR 35
SULFUR COMPOUNDS
SULFUR ISOTOPES
SYNTHESIS
TISSUES
TRITIUM COMPOUNDS
VERTEBRATES