Interaction of spin-labeled analogues of 1,10-phenanthroline and lodoacetamides with Horse Liver Alcohol Dehydrogenase

J. E. Spallholz, L. H. Piette

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


A paramagnetic analog of 1,10-phenanthroline has been synthesized which binds to Horse Liver Alcohol Dehydrogenase forming a stoichiometric 2:1 complex. The compound, 1,10-phenanthroline-5-acetamido-2,2,5,5-tetramethylpyrrolidine-1-oxyl was formed from 5-amino-1, 10-phenanthroline and the acid chloride of 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl. The compound is competitive with NAD+ with an apparent Kd = 2.7 μm. The Electron Spin Resonance absorption of the compound exhibits a strongly immobilized spectrum with a maximum splitting of 66 gauss indicating that the label is located in a highly restricted environment most likely bound to two of the four zincs present in LADH. NADH and 1,10-phenanthroline prevent binding of the paramagnetic analog to the enzyme. A low zinc (approximately two zincs remaining) and a zinc-free LADH analog do not bind the label indicating that the most easily dissociable zincs in the native enzyme are those to which the label binds and are probably catalytically important. Two sulfhydryls presumably associated with enzyme activity have been labeled with two iodoacetamide spin labels with varying chain lengths. The Electron Spin Resonance spectra obtained from these labels indicate that like zinc, these thiols also exist in restricted environments. ESR spectra, stoichiometry of binding and remaining catalytic activity of the spin-labeled LADH suggests that the two catalytically important thiols are in structurally different environments. Inhibition of LADH with the iodoacetamide spin labels reduces the stoichiometry of binding of the paramagnetic analog of 1,10-phenanthroline. These results are suggestive of a close proximity of the zinc and sulfhydryl moieties within the enzyme's active site.

Original languageEnglish
Pages (from-to)596-606
Number of pages11
JournalArchives of Biochemistry and Biophysics
Issue number2
StatePublished - Feb 1972


Dive into the research topics of 'Interaction of spin-labeled analogues of 1,10-phenanthroline and lodoacetamides with Horse Liver Alcohol Dehydrogenase'. Together they form a unique fingerprint.

Cite this