NMR Based Study Of The Interaction Between Human Serum Albumin And Ligands

Human serum albumin (HSA) is the most abundant protein in the circulatory system, synthesized in the liver and secreted into blood stream. The content of HSA is about 60 percent in human serum, and the concentration is about 50 mg/mL (?600?M) in serum for a health adult. The principal function of HSA is to transport a wide variety of drugs and endogenous compounds. HSA consists of 585 amino acids with three homologous domains. Each domain consists of two sub-domains. Since it has many binding sites on the surface, HSA can impact the drugs'delivery and efficacy.HSA is also the most important physiological transporter of essential metal ions such as Cu2+, Zn2+ in the bloodstream. Zinc is not only required for hundreds of essential extra- and intracellular proteins and enzymes but also recruited by toxins such as anthrax lethal factor and staphylococcal enterotoxin. Most of the zinc is bound to HSA in blood plasma. So it is necessary to understand the relationship between zinc and human serum albumin.Crystallization is the most common method to analyze the protein construction and binding sites. But it takes a long time to prepare the samples and the process is very complicated. As a non-destructive technique, Nuclear magnetic resonance (NMR) is widely used to identify the binding sites in solution, for drugs with different affinity and multiple binding sites. No sophisticated sample preparation is required. Therefore, it may not destroy the integrality of sample. But it is difficult to get the information of the binding sites without isotope labeling, since the line width of the 1H NMR spectra for macromolecules, such as HSA, is much broad and the signals are serious overlap.(1) A new spectral editing combine RD-WaterLOGSYwith T2 weighted experiment (T2W-RD-WaterLOGSY) have been applied, it is found that the method can simplify the NMR spectra of HSA efficiently. With the help of the simplified spectra, we can identify the binding sites on HSA continently.(2) T2W-RD-WaterLOGSY method has been used to examine the interaction between ions and HSA. By examining the spectral changes with the alteration of the pH value of the solution. We found that the chemical shift of specific resonances changed dramatically with a rates proportional to pH. Therefore, we can use the relative chemical shift changes of the specific resonance to determine the changes of pH in solution. Furthermore, we found that there is a linear relationship between the intensity of other specific resonance and the zinc concentration. These results indicate that the method may be used to study the interaction of HSA and metal ions.(3) T2W-RD-WaterLOGSY method has been used to examine the interaction between drugs and HSA. By observing the signals change with the increase of drug concentration, we may analysis the affinity and binding site of specific drugs. When drugs binding to Site I, the chemical shift or intensity of some specific resonances were change.(4) Using the method, we can directly detect the HSA in blood serum.