TY - JOUR
T1 - Circular dichroism assay for decarboxylation of optically pure amino acids
T2 - Application to ornithine decarboxylase
AU - Brooks, Harold B.
AU - Phillips, Margaret A.
N1 - Funding Information:
Funding for this research has been provided to M.A.P. by NIH Grant R01 AI34432 and the Welch Foundation Grant I-1257. We thank Andrei L. Osterman, Lisa N. Kinch, and Josep Rizo for helpful discussions.
PY - 1996/7/1
Y1 - 1996/7/1
N2 - A circular dichroism (CD) assay for decarboxylation of optically pure amino acids is described. The viability of this assay is demonstrated using the Trypanosoma brucei ornithine decarboxylase (ODC)-catalyzed reaction of L- ornithine to putrescine and CO2. The results from the CD assay (k(cat) of 7.5 ± 0.7 s-1 and K(m) 230 ± 60 μM) were identical to the results obtained from the commercially available dye-linked assay which couples CO2 production with NADH oxidation (k(cat) of 7.3 ± 0.5 s-1 and K(m) 320 ± 30 μM). The CD assay has advantages over the currently used 14CO2 and dye- linked assays since it can be continuously monitored and does not contain additional enzymes. The CD assay will enable the determination of the effects of pH, ionic strength, and D2O on catalysis by ODC. Furthermore, the availability of cuvets with pathlengths from 0.01 to 100 mm provides an effective range for the CD assay from 10 μM to 2.5 M L-ornithine concentration for this assay. This technique should be generally applicable for steady-state analysis of other decarboxylases but is not easily amenable to the analysis of crude enzyme preparations.
AB - A circular dichroism (CD) assay for decarboxylation of optically pure amino acids is described. The viability of this assay is demonstrated using the Trypanosoma brucei ornithine decarboxylase (ODC)-catalyzed reaction of L- ornithine to putrescine and CO2. The results from the CD assay (k(cat) of 7.5 ± 0.7 s-1 and K(m) 230 ± 60 μM) were identical to the results obtained from the commercially available dye-linked assay which couples CO2 production with NADH oxidation (k(cat) of 7.3 ± 0.5 s-1 and K(m) 320 ± 30 μM). The CD assay has advantages over the currently used 14CO2 and dye- linked assays since it can be continuously monitored and does not contain additional enzymes. The CD assay will enable the determination of the effects of pH, ionic strength, and D2O on catalysis by ODC. Furthermore, the availability of cuvets with pathlengths from 0.01 to 100 mm provides an effective range for the CD assay from 10 μM to 2.5 M L-ornithine concentration for this assay. This technique should be generally applicable for steady-state analysis of other decarboxylases but is not easily amenable to the analysis of crude enzyme preparations.
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U2 - 10.1006/abio.1996.0274
DO - 10.1006/abio.1996.0274
M3 - Article
C2 - 8660610
AN - SCOPUS:0030201077
SN - 0003-2697
VL - 238
SP - 191
EP - 194
JO - Analytical biochemistry
JF - Analytical biochemistry
IS - 2
ER -