PubMed · 2827237
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H Sayo. 1987. [Spin immunoassay].. https://pubmed.ncbi.nlm.nih.gov/2827237/
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Diethyl-dithiocarbamate (DETC) chelating ferrous iron is used during EPR detection of nitric oxide (.NO) as a precursor of spin traps for the quantification of released .NO both in vitro and in vivo. This method is based on the high affinity of water insoluble iron-dithiocarbamate complexes towards .NO. The nitrosyl-iron-dithiocarbamate complex NOFe(DETC)2 is formed with characteristic triplet EPR spectrum. In the present paper we have studied the reaction of water soluble Fe(II)-proline-dithiocarbamate complex, Fe(PDTC)2, with nitric oxide using both EPR spectroscopy and laser flash photolysis. It has been found that the treatment of the Fe(PDTC)2 complex either by authentic .NO dissolved in aqua buffer solutions or by different .NO donors results in the quantitative appearance of an EPR signal of a stable mononitrosyl complex NOFe(PDTC)2 with a g-factor giso = 2.04 and a hyperfine splitting constant aN = 12.5 G. The laser flash photolysis method has been used to study the kinetics of .NO trapping by Fe(PDTC)2. Nitric oxide release from a light sensitive .NO donor, piridazinofuroxan, was induced by a short flash of UV light generated by a XeCl eximer laser. The rate constant of the reaction of .NO with Fe(PDTC)2 was found to be (1.1 +/- 0.3).10(8) M-1s-1, manifesting a high efficiency of .NO trapping by Fe(PDTC)2.
Ribonucleotide reductase catalyzes a key step in DNA biosynthesis and repair, supplying the cell with the four common deoxyribonucleotides. It is thus the target of antiproliferative agents. The enzyme consists of two subunits named protein R1 and protein R2. R1 provides the sites for the nucleotide substrates and redox-active cysteines required for catalysis. R2 harbors a tyrosyl radical essential for activity. We show here that 2'-deoxy-2'-mercaptouridine 5'-diphosphate, a substrate analog, is a very efficient inactivator of ribonucleotide reductase (Ki = 35 microM, Kinact = 0.18 s-1). Inactivation is due to specific scavenging of the protein R2 tyrosyl radical. This unique feature sets this compound apart from other mechanism-based inhibitors such as 2'-azido-or 2'-chloro-2'-deoxyribonucleotide which induce partial or total protein R1 inactivation. During reaction, a transient organic radical was detected by EPR spectroscopy. Its g anisotropy (gz = 2.0620, gy = 2.0265, and gx = 2.0019) and its hyperfine structure are consistent with a perthiyl RSS. radical. The loss of the hyperfine structure by deuterium labeling of the beta protons of R1 cysteines unambiguously shows that the perthiyl radical is located on protein R1. We thus conclude that inactivation of ribonucleotide reductase by 2'-deoxy-2'-mercaptouridine 5'-diphosphate is due to an irreversible transfer of the radical located on protein R2 to a cysteine residue of protein R1.
This study investigates the effect of enzymatic modifications of the HDL(3) surface lipid composition on their physical properties. Human HDL(3) (d: 1.125-1.21 g/ml) was treated either by an exogenous phospholipase A(2) from Crotalus adamanteus or by a sphingomyelinase from Staphylococcus aureus in the presence of albumin for various periods of time in order to obtain several degrees of hydrolysis. Glycerophospholipid hydrolysis ranged from 13 to 81% and sphingomyelinase action led to a 31-92% sphingophospholipid degradation. Physical properties of the surface of HDL(3) were examined by two spectroscopic methods: fluorescence polarisation and electron spin resonance. Glycerophospholipolysis treatment of HDL(3) enhanced the fluorescence anisotropy values (6-18%) and both relaxation correlation time (30-100%) and degree of order. All these results indicated a more rigid environment, a decreased mobility and an increased order of the surface lipids. Conversely, treatment of the HDL(3) with sphingophospholipase induced a progressive fluidization: fluorescence polarisation and degree of order decreasing down to 10% and relaxation correlation time down to 35% compared to native HDL(3). Taken together, all these observations suggest the relative importance of the two major phospholipids to modulate the fluidity and order of the surface of HDL(3) and could account for several recent physiological observations.