Book of Abstracts: Albany 2007
June 19-23 2007
Interaction of non-enzymatically glycated HSA with polyribonucleotide ligands
Extracellular RNA molecules have been detected in human blood, and their level increases with the development of a number of pathological processes (1). The presence of ribonucleic acids in ribonuclease-rich medium can be explained by complex formation of RNA molecules with specific serum proteins. It has been shown earlier that human serum albumin (HSA) is one of the major polyribonucleotide-binding proteins of human blood plasma (2).
Recent reports documented HSA interaction with DNA and RNA (3, 4). Two binding sites with strong and weak association constants were detected for HSA-DNA complex, while the presence of one affinity binding site was demonstrated for albumin complex with RNA. However, the albumin fraction in blood serum is known to be heterogenic, with 6-15% of HSA being non-enzymatically glycated, and the level of glycoalbumin (gHSA) increases in the case of a number of diseases. Unfortunately, the data on the efficiency of nucleic acids binding by this albumin form are absent. We now examine the interaction of various polynucleotide ligands with glycated HSA under physiological conditions.
A sample of glycoalbumin has been prepared in vitro under the conditions described in (5). The fluorescence characteristics of the obtained protein are similar to those of in vivo glycated HSA (6). According to MALDI-TOF MS, the obtained gHSA contained about 15 units of glucose per molecule. As it has been reported earlier (7), up to 15 glucose molecules are condensed on serum albumin in blood of patients with badly-controlled diabetes mellitus. Thus, the structure of gHSA obtained in the present study is similar to that of the in vivo protein state under the pathological condition.
In order to determine the dissociation constants of the protein-polynucleotide complexes, analyze the sequence preference and changes in the biopolymers secondary structure under the complex formation, the methods of fluorescence titration, affinity capillary electrophoresis and circular dichroism were employed. The comparative analysis of poly(A) binding to native and glycated albumins have been carried out using affinity capillary electrophoresis. It has been demonstrated that the gHSA binds poly(A) more efficiently that unmodified HSA (Kd = 0.7 x 10-5 M and 1.3 x 10-5 M, respectively), which confirm our previous results using poly(A)-immobilized affinity columns (2). Interaction of the proteins with poly(A) stabilizes the polynucleotide secondary structure, as shown by circular dichroism.
The data of fluorescence titration (excitation 365 nm, emission 440 nm) of gHSA with polynucleotide ligands demonstrate that the affinity of serum albumin towards nucleic acids depends on the nature of the heterocyclic bases and sugar moiety. Polyriboadenylic acid is bound to gHSA about three times more efficiently than to polyd(A). gHSA affinity towards polypyrimidine sequences (poly(U), poly(C)) is about five times lower than that towards poly(A). At the same time, the dissociation constant of gHSA complex with poly(A) ⋅poly(U) duplex is only slightly more than that of protein-poly(A) complex.
Our results provide the first data regarding the interaction of glycated albumin with polynucleotides, which may help to elucidate the nature and function of extracellular protein-nucleic complexes in human blood in health and disease.
This work was supported by RFBR grant ? 05-04-48447 and SBRAS integration project ?60-2006.
References and Footnotse
1Institute of Chemical Biology &