THIOL-INDUCED OLIGOMERIZATION OF ALPHA-LACTALBUMIN AT HIGH-PRESSURE

Denaturation and aggregation of alpha-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, ge l-permeation chromatography, sodium dodecyl sulfate and gel electropho resis. It was found that the unfolding of alpha-lactalbumin at high pr essure is reversible even in basic pH and at a protein concentration a s large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregat ion of alpha-lactalbumin at high pressure increases significantly in t he presence of low-molecular reducing agents such as cysteine, 2-merca ptoethanol, and dithiothreitol. Maximal yield of alpha-lactalbumin oli gomerization (over 90%) was achieved in the presence of cysteine at th e molar cysteine/protein ratio q=2 and at pH8.5. Apparent molecular we ight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varying pH and red ucing agent. The size distribution shifts in the direction of very lar ge, poorly soluble particles when pH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH5.5 when cysteine (q=2) is used as reducing agent. The oligomers of alpha-lactalbumin ar e stabilized mainly by nonnative interchain disulfide bridges. Circula r dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermol ecular beta-sheets.