The mechanisms underlying the effect of alpha-cyclodextrin on the aggregation and stability of alcohol dehydrogenase.

High concentrations of proteins and enzymes have to be stored for extended periods of time. Under such conditions, at least three major factors contribute to aggregation and loss of protein function: hydrophobicity, propensity to form non-native beta-sheet structure and net charge of the polypeptide chain. Here we evaluate these thermal aggregation factors for horse liver ADH (alcohol dehydrogenase) and the effect of alpha-CyD (alpha-cyclodextrin) on the ADH aggregation, by using fluorescence, CD, UV-visible spectrophotometry, the DLS (dynamic light scattering) technique and the enzymatic activity assay. In addition, we propose the relative importance of the hydrophobic effect on the ADH aggregation. Although ADH readily forms aggregates at higher temperatures, alpha-CyD effectively diminishes this phenomenon. This reduction can be attributed to the prevention of the appearance of larger-size aggregated molecules and also to the higher homogeneity of the small nuclei under the alpha-CyD effect. The observed re-aggregation upon the addition of alpha-CyD/phenylalanine can be attributed to the competition binding of phenylalanine to the internal hydrophobic cavity of alpha-CyD. This signifies that aromatic amino acids are important regional components of the residual structure that may form nuclei for aggregation. The results of dynode voltage changes indicate that the thermal unfolding of ADH is accompanied by protein aggregation, which subsequently leads to irreversible thermal unfolding. Moreover, alpha-CyD causes thermal stabilization and delays the onset of secondary structural unfolding and aggregation by approx. 10 degrees C and the midpoint ('melting') temperatures (T(m)) by more than 5 degrees C. Furthermore, alpha-CyD diminishes the deactivation of the enzyme, decreasing the deactivation constant by more than 50%, and clearly reveals the stabilization of the enzyme not only structurally but also kinetically at higher temperatures.

Alginate as an antiglycating agent for human serum albumin.

Hyperglycemia and the accumulation of advanced glycation endproducts (AGEs) in tissues and serum have important roles in diabetic complications. Therefore, the identification of anti-glycation compounds is attracting considerable interest. In this study, the interaction of human serum albumin (HSA) with fructose, in the absence and presence of alginate, was studied by circular dichroism, absorbance and fluorescence techniques. The characterization study of AGEs was performed using autofluorescence, fibrillar formation, the increase in absorbance and the quantification of free lysine side chains. The results indicate that alginate inhibits the fructation of HSA as observed by a reduction in the formation of fluorescent AGEs and fibrils. Furthermore, alginate reduces the amount of modified lysine side chains, signified by the lack of increase in absorbance, and increases the helicity of this protein.

Formation of the molten globule-like state during prolonged glycation of human serum albumin.

The interaction of reducing carbohydrates with proteins leads to a cascade of reactions that are known as glycation or Maillard reaction. We studied the impact of incubation of human serum albumin (HSA) with glucose, at various concentrations and incubation times, on the extent of HSA glycation and structural changes using circular dichroism (CD), fluorescence, and microviscometer techniques. The number of moles of glucose bound per mole of HSA (r), the number of reacted lysine and arginine residues, and the Amadori product formation during glycation were determined using 3-(dansylamino) phenyl boronic acid, fluorescamine, 9, 10 phenanthrenequinone, and p-nitroblue tetrazoliumchloride, respectively. The formation of advanced glycation end products (AGE) was detected using the autofluorescence characteristic of samples. We identified three stages of Maillard reaction for HSA upon incubation with the physiological level of glucose (0-630 mg/dl): the early, intermediate and late stages, which occurred after 7-14, 21, and >28 days of incubation, respectively. Structural information, Stokes radius, and 1-anilinonaphthalene-8-sulfonate (ANS) binding data indicated the formation of a molten globule-like state of HSA after 21 days of incubation with 35 mM (630 mg/dl) glucose. Thus, the extent of the Maillard reaction was influenced by the concentration of glucose and incubation time, such that longer exposure of HSA to glucose may have a more deleterious effect on its structure and especially on its half-life and turnover in the circulation. Our results suggest that in acute diabetes mellitus patients, HSA, after 21 days of glycation, passes through a molten globule-like state and may contribute to the pathogenesis of diabetes, and perhaps other diseases.

Biological activity of some monocyclic- and bicyclic beta-lactams with specified functional groups.

This Review contains our recent studies on evaluation of biological activities associated with monocyclic beta-lactams and bicyclic beta-lactam antibiotics containing various heteroatoms. A series of bicyclic beta-lactams was synthesized, which possessed electron-withdrawing groups, such as an ester, mesylate, and triflate functionality. These beta-lactams exhibited enhanced antibacterial activity.