Spectroscopic and Molecular Modeling Studies on Binding of Fleroxacin with Human Serum Albumin.

Fleroxacin (FLRX) is a new member of the class of fluoroquinolones, its effects on human serum albumin (HSA) and the mechanism of action are poorly understood, Especially, the secondary structural alterations of HSA induced by FLRX and the inner filter effect, which resulted in a spurious decrease in the observed fluorescence intensity and affected the binding parameters calculated from it are not considered. In this paper, binding of FLRX to HSA has been studied using multi-spectroscopy and molecular modeling methods. Fluorescence spectra revealed that the observed fluorescence quenching of HSA by FLRX was due to a 1∶1 complex formation by a static quenching process with a constant of 105 L·mol-1. The thermodynamic parameters (ΔH and ΔS) were calculated to be -107.99 kJ·mol-1 and -240.99 J·mol-1·K-1 via the Van't Hoff equation, which indicated that hydrogen bond and van der Waals force were the dominant intermolecular force. From the synchronous fluorescence, FT-IR and three dimensional fluorescence spectra, it was evident that the binding of FLRX to HSA induced a conformational change in the protein, and the alterations of secondary structure were quantitatively calculated by the evidence from FTIR spectra with reductions of α-helices of about 18.3%, decreases of ß-sheet structures of about 9.6%, and increases of ß-turn structures of about 18.0%. Site marker competitive experiments showed that phenylbutazone and FLRX shared a common binding site Ⅰ corresponding to the subdomain Ⅱ A of HSA. The binding details between FLRX and HSA were further confirmed by molecular docking studies, which revealed that FLRX was bound at subdomain Ⅱ A through multiple interactions, such as hydrogen bond, hydrophobic and van der Waals, etc. The accurate and full basic data in the work is beneficial to clarify the binding mechanism of FLRX with HSA and is helpful for understanding its effect on protein function during the blood transportation process.

[Detection of Adulteration in Milk Powder with Starch Near Infrared].

Three China trademarks of milk powder called Mengniu, Yili, Wandashan were taken as testing samples. Each of them mixed varied amount of starch in different gradient, which were consisted of 32 adulterated milk powder samples mixed with starch, was taken as standard samples for constructing predicted model. To those 32 samples, the reflecting spectrum characteristics in middle wave of near infrared spectrum with Near Infrared Spectrum Analyzer (Micro NIR 1700) produced by JDSU Ltd. USA were collected for five repeats in five different days. The time span was nearly two months. Firstly, we build the model used the reflecting spectrum characteristics of those samples with biomimetic pattern recognition (BPR) arithmetic to do the qualitative analysis. The analysis included the reliability of testing result and stability of the model. When we took ninety percent as the evaluation threshold of testing result of CAR (Correct Acceptance Rate) and CRR (Correct Rejection Rate), the lowest starch content of adulterate milk powder in all tested samples which the tested result were bigger than that abovementioned threshold was designated CAR threshold (CAR-T) and CRR threshold (CRR-T). CAR means the correct rate of accepting a sample which is belong to itself, CRR means correct rate of refusing to accept a sample which is not belong to itself. The results were shown that, when we constructed a model based on the near infrared spectrum data from each of three China trademark milk powders, respectively, if we constructed a model with infrared spectrum data tested in a same day, both the CAR-T and CRR-T of adulterate starch content of a sample can reach 0.1% in predicting the remainder infrared spectrum data tested within a same day. The three China trademarks of milk powder had the same result. In addition, when we ignored the trademarks, put the spectrum data of adulterate milk powder samples mixed with the same content of starch of three China trademarks milk powder together to construct a model, the CAR-T of mixed starch content of a sample may reach 0.1%, the CRR-T can reach 1%, if the model construction and predicting were performed with near infrared spectrum data tested in a same day. However, the CAR-T can just stably reach up to 5% and the CRR-T have the same result, if the model construction and predicting were crossly performed with mixed near infrared spectrum data tested in different days. Furthermore, the correct recognizing threshold mixed starch of a sample can stably reach up to 1% and the CAR-T can reach 5%, if the model construction was based on near infrared spectrum data combined the previous four days to predict the output of the another day. On the other hand, we also engaged quantitative analysis to the starch content in milk power with two kinds of arithmetic (PLSR, LS-SVR). In contrast with the testing outputs, the reliability of both the CAR-T and CRR-T in qualitative analysis was further validated.

[Study of interaction between levofloxacin and human serum albumin by multi-spectroscopic and molecular modeling methods].

Levofloxacin (LVFX) is widely used in clinical treatment due to it has a broad spectrum of in vitro activity against Gram-positive and Gram-negative bacteria. Human serum albumin (HSA) is the most abundant protein in plasma and constitutes approximately half of the protein founds in human blood. And more than 90% of the drugs used in people are bound to HSA. So it is commonly used for the investigation of drug-serum albumin interaction because the binding will significantly influence the absorption, distribution, metabolism excretion, stability and toxicity of the drugs. Therefore, detailed investigating the interaction of LVFX with HSA is very important to understand the pharmacokinetic behavior of the LVFX. In this paper, the interaction of LVFX and HSA has been studied fluorescence, UV, Fourier transform infrared (FT-IR) and molecular modeling method. The results indicated that LVFX induced the intrinsic fluorescence quenching of HSA though a static quenching procedure, and the effective binding constants (K(a)) were calculated to be 9.44 x 10(4) L x mol(-1) (294 K) and 2.74 x 10(4) L x mol(-1) (310 K) by used of the Stern-Volmer equation. According to the Vant's Hoff equation, the reaction was characterized by negative enthalpy (deltaH = -59.00 kJ x mol(-1)) and negative entropy (delta S = - 105.38 J x mol(-1) x K(-1)), indicated that the predominant forces in the LVFX-HSA complex were hydrogen bonding and van der Waals forces. By displacement measurements, the specific binding of LVFX in the vicinity of Site I of HSA was clarified. The binding distance of 3.66 nm between Trp214 and HSA was obtained by the Förster theory on resonance energy transfer. Furthermore, the binding details between LVFX and HSA were further confirmed by molecular docking studies, which were consistent with the experimental results. The alternations of protein secondary structure were calculated from FT-IR spectra. Upon formation of LVFX-HSA complexes, the amount of alpha-helical structures were decrease, but the numbers of beta-sheet structures, beta-turn structures and random structures were increase, respectively. This result indicated that LVFX induced unfolding of the polypeptides of HSA.

Determination of sparfloxacin concentrations in chicken serums and residues in chicken tissues and manures using self-ordered ring fluorescence microscopic imaging technique.

Based on the self-ordered ring (SOR) fluorescence microscopic imaging technique on a hydrophobic glass slide with Zn2+ and cetyltrimethyl ammonium bromide (CTMAB) as sensitizer, and poly (vinyl alcohol) 124 (PVA-124) and NH3-NH4Cl (pH 10.00) as the medium, a method has been developed for determining sparfloxacin (SPFX) concentrations in chicken serum and residues in chicken tissues and manures. When the droplet volume was 0.20 microL, SPFX was determined in the range of 1.38 x 10(-13)-2.03 x 10(-12) mol x ring(-1) (or 6.90 x 10(-7)-1.02 x 10(-5) mol x L(-1)), and the limit of detection (LOD) was 14 fmol x ring (or 6.90 x 10(-8) mol x L(-1)). The recoveries of SPFX at all different spiked levels are in the range of 90.74%-106.61% when the methanol or acetonitrile were used as extracting agent, respectively, and the relative standard deviations (RSDs) are less than 3.0%. This study expands the applied fields of SOR technique in drug concentrations and residues determination.