生物物理化学.pptx

1、pH Effect on Protein G Orientation on Gold Surfaces andCharacterization of Adsorption Thermodynamics,陈晓宇2014101928,1、ABSTRACT,2、INTRODUCTION,Protein adsorption,Ionic strength,pH值,cosolvent,Surface topography,pH值,3、METHODS,1、Quartz Crystal Microbalance(QCM)：石英晶体微天平,2、X-ray Photoelectron Spectroscopy(

2、XPS)X：射线光电子能谱,4、RESULTS AND DISCUSSION,Figure 1. (A) Typical binding experiment shows proG binding response to a bare Au surface (1), a PBS rinse step (2), the antibody binding response to the adsorbed proG (3), the antigen binding response to the immobilized antibody (4), and a nal PBS rinse (5). I

3、ncluded is a negative control in which no binding analyte was used, which shows that the resonant frequency remains constant (standard deviation 0.4 Hz) in the absence of protein.,1、Typical Binding Experiment.,Figure 1.(B) Binding responses shown for proG, BSA, and anti-E. coli adsorption to bare Au

4、. All three remained adsorbed when ow was switched back to PBS over a three hour period. ProG, BSA, and antibody caused f = 14.2 1.1, 11.7 1.2, and 16.7 3.0 Hz, respectively (n = 8).,2、Adsorbed Protein G Saturates the Au Surface.,3、Presence of Adsorbed Protein G Veried Using XPS.,Figure 2. (A) XPS s

5、pectra of a clean Au surface showed a single Au 4p3/2 peak at 547 eV and no nitrogen (N) 1s or oxygen (O) 1s peak.XPS spectra of proG-exposed Au showed clear presence of adsorbed protein by appearance of the N 1s the O 1s peaks at 399 and 533 eV , respectively, as well as reduction in Au 4p3/2 peak

6、height.,4、Protein G Saturates the Au Surface Regardless of pH.,Figure 2. (B) ProG adsorption to Au gives nearly identical fractional coverage regardless of the pH . The net shift in all cases was 14.7 1.9 Hz (n = 3). Over the course of one hour, a control in which no proG was introduced showed no sh

7、ift (0.4 Hz). Rapid increase and decrease dynamics at t= 5 and 50 min, respectively, are due entirely to density dierences between the acetate-prepared proG solutions and buers.,5、Protein G Orientation Is Aected by pH.,Figure 3. (A) Binding of anti-BSA at a single pH (7.2) to proG which was adsorbed

8、 at pH = 7.2, 5.0, and 3.0 shows noted dierences in the amount of bound antibody depending on the pH at which the proG was immobilized. Antibody binding to the proG adsorbed at the highest pH (7.2) gave the highest response, while binding to the proG adsorbed at lower pH (5.0 and 3.0) gave 37% reduc

9、tion in antibody binding.,6、pH Eect on Protein G Binding Orientation Confirmed by a Second Antibody-Antigen System.,Figure 3. (B) Response of anti-E. coli at a single pH (7.2) to proG adsorbed at pH 7.2, 5.0, and 3.0 shows the same trend of proG orientation as found using the anti-BSA system. Result

10、s indicate that the highest amount of properly oriented proG was obtained when absorbed at near-neutral pH.,7、Discussion on pH Eects on Protein G Orientation.,Figure 4. (A) Schematic showing the pH eect on the orientation of the Fc binding region (the C23 domain) of proG.,8、Protein G Binding Energet

11、ics to Au Suggest Chemisorption-Like Binding Mechanism.,Figure 4. (B) Adsorption of proG at 296, 303, and 308 K shows that the equilibrium amount of adsorbed protein decreases with increased temperature, indicating an exothermically and entropically driven adsorption process.,5、CONCLUSION,Solution pH signicantly aects adsorbed protein G orientation on Au. The results indicate that pH = 7.2 exhibited the most properly oriented protein G in two dierent antibodyantigen systems (BSA and E. coli O157:H7). The mechanism of the Au-protein adsorption process was characterized by