Temperature dependence of human a1-acid glycoprotein behavior induced by mixed solvents and acid pH


L. Maršálková1, V. Kopecký Jr.2, V. Karpenko1


1 Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Albertov 6, CZ-12843 Prague 2, Czech Republic; marsalko@natur.cuni.cz

2 Institute of Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 5, CZ-12116 Prague 2, Czech Republic


Orosomucoid is one of the original names of α1-acid glycoprotein (AGP throughout this paper), which is a protein from the α1-globuline fraction of human blood serum [1] classified among the superfamily of lipocalins. Its molecule contains 183 amino acids with 21 possible substitutions in one polypeptide chain linked by two disulfide bonds. The content of carbohydrate moiety is slightly over 40% of the total molecular weight [1] of 41 kDa of AGP. Five branched carbohydrate units terminated by sialic acid residues are linked to asparagine residues of the peptide chain by the N-glycosidic bond. The isoelectric point of AGP has been found to vary between pH 1.8 and 2.7, depending on the buffer chosen. The native structure of AGP is characterized by a low α-helix and a high β-sheet [2] content. Although AGP has been studied for over fifty years and successfully crystallized already in 1959, all attempts at its X-ray analysis have failed; there is only a 3D computer model [3] of the peptide part of the AGP molecule based on molecular and homology modeling. AGP is a glycoprotein occurring naturally in human blood serum; it is well-known that its blood level increases under some pathological conditions, but the exact role of this protein is not entirely clear.

This glycoprotein has been studied in our laboratory in the basic pH range in both aqueous and mixed solvents, whereas in the acid region the investigation has been done only in aqueous solution [4]. We have chosen the acid pH region for our study because of low isoelectric point of AGP; stability of protein molecules in the vicinity of this pH is of particular interest. As mixed solvent mixtures water – low aliphatic alcohol have been selected. It can be assumed that alcohols exert many-sided effect on protein molecule as well as on the solvent, water. Decrease in permittivity of a solution due to the alcohol results in enhancing of all electrostatic interactions in the system, not only of inter- and intramolecular, but it also concerns the binding of inorganic ions present in the solution. Not less important is the effect of alcohol on the structure of water and on the hydration shell of the protein molecule. With current knowledge of water structure, this effect cannot be quantified. 

We have been interested in figuring out the temperature changes of AGP molecule in presence of the two lowest aliphatic alcohols – methanol, and ethanol in the acid region. Weight fraction of alcohols was 0.20 over whole pH range studied (pH 2–6) except for pH 3. At this pH it was necessary to lower weight fraction of methanol to 0.01, and ethanol to 0.10, respectively. Temperature measurements consisted of three steps: heating of the sample from 25 °C to 80 °C, cooling it back on 25 °C, and heat it again on 80 °C. The behavior of AGP has been studied using UV-VIS absorption spectroscopy, fluorescence spectroscopy and spectroscopy of circular dichroism. Spectral sets of each spectroscopic method used (at desired pH or at an aliphatic alcohol) have been examined by principle component analysis. Concerning statistical methods, two-dimensional (2D) correlation spectroscopy was applied as well as a new method of a multivariate statistics which provides the specific order of the spectral intensity changes taking place during the measurement on the value of variable affecting the spectra. Moreover, heterospectral 2D correlation spectroscopy enables joint data evaluations of spectral sets measured by different spectroscopic methods at the same conditions. Finally, it has been demonstrated that in presence of both studied alcohols AGP has the lowest solubility in the narrow region around pH 3. Cyclic temperature measurement made it possible to follow course of structural changes in the molecule from the point of view of their reversibility. Our experiments have shown pronounced influence of both temperature and pH on the thermal stability of AGP molecule in the aqueous solution of alcohols.


Ministry of Education of the Czech Republic and the Grant Agency of the Czech Republic are gratefully acknowledged for support (No. MSM 0021620835, No. 202/06/P208, respectively).


1.     Schmid K. in: Alpha1-Acid Glycoprotein: Genetics, Biochemistry, Physiological Functions, and Pharmacology (P. Baumann et al., ed.), p.7, Alan R. Liss Inc. 1989.

2.     Kodíček M., Infanzón A., Karpenko V.: Biochim. Biophys. Acta 1246, (1995), 10.

3.     Kopecký V. jr., Ettrich R., Hofbauerová K., Baumruk V.: Biochim. Biophys. Res. Commun. 300, (2002), 41.

4.     V. Karpenko, J. Horálková, M. Kodíček, Collect. Czech. Chem. Commun., 62, (1997), 1533.