Human serum albumin (HSA) acts as a carrier for testosterone, other sex hormones, fatty acids, and drugs. However, the dynamics of testosterone's binding to HSA and the structure of its binding sites remain incompletely understood. Here, we characterized the dynamics of testosterone's binding to HSA and the stoichiometry and structural location of the binding sites using two-dimensional nuclear magnetic resonance (2D NMR), fluorescence spectroscopy, bis-ANS partitioning, and equilibrium dialysis, complemented by molecular modeling. 2D NMR studies showed that testosterone competitively displaced 18-[ 13C]-oleic acid from at least three known fatty acid binding sites on HSA that also bind many drugs. Binding isotherms of testosterone's binding to HSA generated using fluorescence spectroscopy and equilibrium dialysis were nonlinear and the apparent Kd varied with different concentrations of testosterone and HSA. The binding isotherms neither conformed to a linear binding model with 1:1 stoichiometry nor to two independent binding sites; the binding isotherms were most consistent with two or more allosterically coupled binding sites. Molecular dynamics studies revealed that testosterone's binding to fatty acid binding site 3 on HSA was associated with conformational changes at site 6, indicating that residues in in these two distinct binding sites are allosterically coupled. Conclusions There are multiple, allosterically coupled binding sites for testosterone on HSA. Testosterone shares these binding sites on HSA with free fatty acids, which could displace testosterone from HSA under various physiological states or disease conditions, affecting its bioavailability.

ABSTRACT:Software companies face a lot of difficulties in choosing a correct development model when projects have to confront a lot of risk factors ranging from low to high. When software development becomes complex in nature due to risk, companies usual practice is to switch over to the only risk detection traditional model -the Spiral. Spiral model is considered to be the best conventional model for risk analysis. But in modern developmental style, all the development cycles around a single trend -the Agile. The main objective of this paper is to make a study on these two models and to get a conclusion on the impact of risk assessment and its success factors on complex software developmental projects.

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