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Biomedical subjects

R Kluger

Publications and source records attributed to R Kluger.

44 records · Page 3Linked to original sources
PubMed1978

Chromium (III) thiamin diphosphate: a new probe of enzyme coenzyme--metal ion binding. Interaction with wheat germ pyruvate decarboxylase.

Chromium (III) thiamin diphosphate (CrTDP) is a substitutionally inert complex which is a physical and kinetic probe of the binding mode of metal ion and coenzyme in thiamin diphosphate (TDP) dependent enzymes. CrTDP is prepared by reaction of aquated Cr (III) and TDP and purifed by ion-exchange chromatography. CrTDP binds to the apoenzyme of wheat germ pyruvate decarboxylase, giving an inactive holoenzyme. Chromium (III) ion binds to the apoenzyme in a manner that suggests, in light of the results with CrTDP, that there are two metal ion binding sites. Extension of the use of CrTDP to other enzymes can give specific information about binding and can introduce an active site reporter group

Apoenzymes↗
PubMedDate not supplied

Efficient chemical introduction of a disulfide cross-link and conjugation site into human hemoglobin at beta-lysine-82 utilizing a bifunctional aminoacyl phosphate.

The creation of a cross-link containing a disulfide into hemoglobin has been accomplished with a site-directed reagent, N,N'-bis(Cbz-cystinyl)bis(methyl phosphate) (1). This is prepared from the reaction of the bis acid chloride of N-protected cystine with dimethyl phosphate followed by O-demethylation with methyl iodide in acetone. Reaction with deoxyhemoglobin produces two main products: cross-linked hemoglobin as the bis(cystinyl amide) of the epsilon-amino group of the side chain of Lys-82 of the two beta subunits as well as material that has each of the same amino groups modified as the cysteinyl amide but not cross-linked. Addition of 2-mercaptoethanol cleaves the disulfide in the material that is not cross-linked while leaving the disulfide intact in the cross-linked species. Dithiothreitol reduces the disulfide in the cross-linked species as well as in the species that is not cross-linked. Spontaneous oxidation in air converts all of the reduced material to the cross-linked bis(cystinyl amide) of hemoglobin. The reagent permits controlled introduction of cystinyl groups at lysyl residues, leading to formation of sulfhydryl groups by reduction and the possibility of re-forming the cross-links or forming conjugates.

Binding Sites↗
PubMedDate not supplied

Molecular necklaces. Cross-linking hemoglobin with reagents containing covalently attached ligands.

Hemoglobin can be cross-linked and converted to a bioconjugate in one step by a "molecular necklace", a reagent that contains two reacting sites and a pendant ligand. The compound to be conjugated is activated as an electrophile. The activated material is then combined with a reagent (3-aminoisophthalic acid) that contains a nucleophilic (amino) site and two latent (carboxyl) sites. The latent sites of the product are activated as 3,5-dibromosalicylates to produce the cross-linker. Illustrative examples of cross-linking are presented with pendant biotin [bis(3,5-dibromosalicyl) N-biotinyl-5-aminoisophthalate] and pendant N-trifluoroacetyl-L-isoleucylglycine [bis(3,5-dibromosalicyl) N-(N-trifluoroacetyl-L-isoleucylglycyl)-5-aminoisophthalate) ]. The resulting modified hemoglobins contain two principal types of cross-link: (beta-Lys-82-beta'-Lys-82) and (alpha-Lys-99-alpha'-Lys-99). The functional properties of the modified hemoglobin containing biotin in a (beta-Lys-82-beta'-Lys-82) cross-link are (pH 7.4, 55 microM heme, 25 degrees C, 0.1 M chloride, and 50 mM Bis-Tris) P(50) = 4.9 Torr, n(50) = 3.0, values which are approximately the same as for native hemoglobin. The results of affinity chromatography of the biotinylated cross-linked hemoglobin using a column of immobilized avidin indicate that the pendant biotin is much less accessible than free biotin. We suggest that the results are consistent with the pendant species being strongly attracted into the hemoglobin environment.

Binding Sites↗