Domenico Segnini

Evidence of soluble proteins binding adriamycin by affinity chromatography

The presence in various tissues of soluble proteins binding adriamycin is evidenced by affinity chromatography.

Isolation of an urate-binding protein by affinity chromatography

This paper describes an affinity chromatography procedure to purify an urate binding protein from human serum. The specific ligand was 8-amino-2,6-dihydroxypurine bound to Sepharose through the amino group. The specific elution was obtained with an uric acid or allopurinol solution. Electrophoretic analysis of the eluted protein shows a single sharp band with an α2-globulin mobility. Molecular weight, determined by gel filtration, is approximately 70,000 daltons.

Urate-Binding Proteins in Plasma Studied by Affinity Chromatography

The existence in humans of one or more plasma proteins, which specifically bind uric acid (UA) is still discussed. There is no agreement on the nature of the binding protein, identified as the serum albumin1, or as a specific α1-α2-globulin2, nor on the extent of the bound quote. Most investigations have been till now performed by ultrafiltration1 or column chromatography2, but the specificity and the sensitivity of both are rather unsatisfactory; for example, recovery is lesser than 30% in the later technique3. On the other hand, the existence itself of a significant urate binding to human plasma proteins has been denied even recently, by means of continuous ultrafiltration and equilibrium dialysis4.

AFFINITY LABELING OF HISTIDINE AND LYSINE RESIDUES IN THE ADENOSINE-DEAMINASE SUBSTRATE BINDING-SITE

1. Adenosine deaminase was inactivated by 9-(4-bromoacetamidobenzyl)-adenine (I) and 9-(2-bromoacetamidobenzyl)adenine (II), two affinity labels. 2. The stoichiometry of the reaction with reagent II is reported: 1 mol reagent is bound per mol inactive enzyme. Amino acid analysis of the 6 N HCl hydrolyzate of the inactive enzyme identified CM-histidine as the main alkylation product. This is the first evidence of the presence of a histidine in the active site region. 3. The alkylation rate and involved amino acid residues were studied for both reagents I and II, at pH 8 and 5.5. The particular reactivity of a lysine near or in the active site is discussed.

Characterization of an Urate Binding α2-Globulin from Human Serum

Since urate binding to serum protein may influence the deposition in joints and the renal excretion, several authors have studied this interaction with different results1,2.