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The oxidation of NN-dimethyl-p-phenylenediamine by oxidizing agents and by caeruloplasmin.

ABSTRACT: 1. The oxidation of NN-dimethyl-p-phenylenediamine (DPD) by inorganic oxidants and by caeruloplasmin was studied. Some experiments were also made with NNN'N'-tetramethyl-p-phenylenediamine (TPD). 2. E(mM) (550) of the first free radical oxidation product of DPD (DPD(+)) was 9.8 and E(mM) (563) of the corresponding product of TPD (TPD(+)) was 12.5. 3. The non-enzymic decomposition of DPD(+) was studied with respect to temperature, pH, concentration and DPD/DPD(+) ratio, thus defining conditions for enzyme experiments under which DPD(+) extinction at 550mmu was proportional to enzyme activity. 4. Rates of oxidation of DPD to DPD(+) by caeruloplasmin were constant over a range of DPD concentrations. At low DPD concentrations a lag period occurred, which was eliminated by addition of DPD(+). 5. A lag period was not observed with TPD, but at low TPD concentrations the rate of TPD(+) formation was greater when TPD(+) was added. This suggests that TPD(+) may compete weakly as a substrate with TPD and may be oxidized further by the enzyme before a non-enzymic reaction with TPD to form more TPD(+). 6. With DPD sulphate or acetate or TPD sulphate as substrate, Lineweaver-Burk plots were curved. With DPD hydrochloride the chloride ion caused inhibition at higher concentrations, opposing the curvature. 7. Curved Lineweaver-Burk plots were interpreted in terms of two types of substrate binding site with different K(m) values but similar V(max.) values. 8. The apparent thermodynamic changes associated with enzyme-substrate-complex formation at the sites with higher K(m) suggest that considerable conformational change may occur on binding at these sites. 9. With substrate concentrations at which only the low-K(m) sites are involved 2mol. of DPD(+)/mol. of caeruloplasmin are formed before a steady state is established. At higher substrate concentrations up to 3.2mol. of DPD(+)/mol. of caeruloplasmin are formed at this initial stage. 10. Results are discussed in relation to caeruloplasmin structures in which (a) two valence-changing and two permanently cuprous copper atoms are more accessible than the remaining four copper atoms or (b) binding of substrate at one site hinders access of substrate to another site.


PROVIDER: S-EPMC1270408 | BioStudies | 1967-01-01

SECONDARY ACCESSION(S): 10.1042/bj1030289

REPOSITORIES: biostudies

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