A/C DIAGNOSTICS ENZYMATIC L-Cysteine ASSAY
Principle of the A/C Enzymatic L-Cysteine Assay
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The A/C Enzymatic total
CYS assay is based on a four-reagent protocol. Two recombinant enzymes are used
with a DBPDA color reaction quantified in a absorbance or fluorescence reader. The basic version is run in 96-
Step 1. Samples are reduced by DTT to generate free reduced L-CYS and homocysteine (HCY). Simultaneous use of
recombinant s-adenosylhomocysteine hydrolase (rSAHH) with excess adenosine (ado) converts the reduced HCY to
S-adenosylhomocysteine (SAH), and eliminates interference by HCY.
Reduced HCY + Ado Ado-HCY + H20
Step 2. Recombinant methioninase (rMETase) is added to generate H2S from L-cysteine.
Reduced cysteine Pyruvate + H2S + NH3
Step 3. H2S combines with DBPDA to form an absorbent/fluorescent chromogen. Absorbance is read at 675 nm (660-680nm).
DBPDA + H2S 3,7-Bis (dibutylamino)
96 -well plate format
10 or 20 µl sample
Endpoint measurement at 675 nm (660-680nm)
Measurement Range of 16.9 - 600 µmol/L
Inter-assay CV: <4.4 % and Intra-assay CV: < 4.8%.
The assay has no significant interference from lipemic and hemolytic substances.
K3Fe (CN) 6