Ultraviolet (UV) radiation, temperature, and time can degrade proteins. Here, the authors show that gold nanoparticles significantly protect human serum albumin from denaturation when exposed to “stressing” conditions such as UV irradiation and sustained exposure in suboptimal conditions. In particular, the authors show that gold nanoparticles significantly reduce the decrease in secondary structure induced by UV irradiation or extended exposure to ambient temperature.

Ultraviolet radiation, pH, and temperature can destabilize and unfold proteins. It would be extremely useful to prevent this destabilization. One of the most common forms of protein denaturation involves the loss of conformation of native proteins. Circular dichroism (CD) is an excellent and sensitive technique for determining the content of the different secondary structure elements of proteins and to measure their variation under different conditions, such as UV-irradiation, temperature, or pH changes.1–5 Synchrotron radiation circular dichroism (SRCD) spectroscopy relies on the very bright light produced by a synchrotron ring, used as the light source, which results in a higher photon flux over a wider wavelength range than in a conventional CD instrument.6–10 The Diamond B23 SRCD beamline has a highly collimated beam that allows the use of very long pathlength cells (10 cm) with low volume to collect CD spectra of proteins at very low concentrations [as low as 10 nM for human serum albumin (HSA)].1,4 The high flux covers a much wider spectral range than other SRCD beamlines, down to 165 nm.7 However, the high photon flux of the synchrotron radiation may induce protein unfolding,11,12 which can be monitored in real time by CD.13 We have previously shown that gold nanoparticles (AuNPs) can effectively stop this UV-induced unfolding of HSA caused by synchrotron radiation.1 In addition, AuNPs prevent protein aggregation14 and help in refolding thermally denatured proteins when bearing dicarboxylate side chains.15 

Here, we show that citrate-stabilized AuNPs increase the stability of HSA exposed to “stressing conditions,” such as UV irradiation, increase in temperature, and long-term exposure in suboptimal conditions. These results suggest possible applications and benefits of gold nanoparticles in the medical field in addition to their role as novel agents for cancer therapy.16 

The gold nanoparticles were made by first heating 100 ml of a 0.5 mM HAuCl4 solution to 97 °C in a microwave reactor (Discover S by CEM). After 5 min temperature stabilization, 2.5 ml of a 0.1 M solution of sodium citrate was injected with vigorous stirring into the HAuCl4 solution and the reaction mixture was kept at 97 °C for 20 min. The final suspension was cooled down to room temperature. The nanoparticles were concentrated five times by centrifugation at 12 000 g during 25 min at 4 °C with a centrifuge 5430R from Eppendorf (Hamburg, Germany).

HSA fatty acid free, globulin free was purchased from Sigma Aldrich diluted in sodium phosphate buffer (1 mM, pH 7.4). Gold nanoparticles were diluted in the presence or absence of HSA. In presence of HSA, the AuNPs/HSA ratios were: 1/400, 1/1000, 1/5000, or 1/10 000. AuNP-HSA complexes at different nanoparticle–protein ratios were formed by adding variable volume of 15 and 150 μM of the HSA to AuNP concentrated at 2 nM (molarity of particles).

1. Transmission electron microscope

AuNP were visualized using a transmission electron microscope (JEOL 2100, Japan) at an accelerating voltage of 200 kV. The samples were prepared by placing a drop (5 μl) onto ultrathin Formvar-coated 200-mesh copper grids (Tedpella, Inc.) and left to dry in air at 4 °C. For each sample, the size of at least 100 particles was measured to calculate the average and the size distribution. Digital images were analyzed with the imagej software and a macro performing smoothing (3 × 3 or 5 × 5 median filter), manual global threshold and automatic particle analysis provided by imagej. The macro can be downloaded from http://code.google.com/p/psa-macro. The circularity filter of 0.8 was used to exclude agglomerates that formed during drying.

2. Differential centrifugation sedimentation measurement

Differential centrifugation sedimentation data have been collected using a sucrose gradient 8%–24% (w/w) with a CPS disk centrifuge model DC24000 (CPS instruments, Inc.) running at 22 000 rpm.

3. Asymmetric flow field-flow fractionation

HSA-AuNP complexes were purified by asymmetrical flow field-flow fractionation (AF4). The AF4 system used was a Postnova AF2000 equipped with a PN3212 spectrophotometer UV detector (tuned at 525 and 500 nm), and a PN3412 fluorescence detector (excitation wavelength tuned at 280 nm and emission wavelength tuned at 340 nm), all from Postnova (Postnova Analytics, Landsberg, Germany). Sodium phosphate buffer (1 mM, pH 7.4) was used as carrier and cross flow. The channel was set up with a 350 μm spacer and using a regenerated cellulose membrane with 10 kDa cut-off. The detector flow was set at 0.5 ml min−1. The injection step has been done at 0.25 ml min−1 for 5 min. The elution time came after a transition step of 1 min with a cross flow of 2 ml min−1 for 5 min. Then, the cross flow was set up to decrease linearly to 0 ml min−1 in 10 min. The DLS Zetasizer from Malvern was used online with the AF4. The flow cell was a Quartz cuvette ZEN0023 from Malvern. The temperature of analysis was set up at 25 °C and every run lasted 5 s. Samples were collected with a fraction collector from Postnova.

Circular dichroism spectra were collected using either a Jasco CD J810 or a Chirascan CD (Applied Biophysics) instrument. A 1 cm pathlength low volume quartz cell was used to analyze low concentration of AuNP-HSA in complex. For each sample 4–9 scans were acquired and averaged according to the quality of the spectra and the concentration of protein. The CD spectra of free HSA and AuNP-HSA samples were baseline-corrected by subtracting the CD spectrum of the buffer (for free HSA) or the CD spectrum of AuNP at the same concentration than in AuNP-HSA samples (for AuNP-HSA complexes). The baseline-corrected spectra were smoothed with Savitzky-Golay smoothing function of six points. CD spectra were analyzed with the dichroweb analysis software using the continll algorithm and the protein dataset 7 (http://dichroweb.cryst.bbk.ac.uk). Samples for UV-denaturation and thermal unfolding studies were obtained by separating (by AF4) two fractions: the free, unbound HSA, and the AuNP-HSA complexes. Samples used to monitor changes in the secondary structure of HSA in different storage conditions (at 4 and 20 °C) were prepared by mixing HSA with gold nanoparticles without further purification.

SRCD experiments were performed using a nitrogen-flushed module B end-station spectrophotometer at the B23 synchrotron radiation CD beamline at the Diamond Light Source (Oxfordshire, UK). Measurements were carried out at 23 °C unless otherwise stated with wavelength set between 185 and 260 nm, 1 mm slit equivalent to 1.5 nm bandwidth, increment of 1 nm, and interval time of 1 s. Data from UV denaturation experiments were collected on protein samples as repeated scans, without any averaging over 30 scans. The cell used has a 10-cm pathlength and a volume of 820 μl. Protein secondary structure was estimated with the B23 software, cdapps, available for in-house and remote processing cdapps (Ref. 17) using the contin-ll algorithm and the dataset CLSTR.

For thermal unfolding studies, HSA were incubated in the presence and absence of AuNP at 20 °C and spectra were recorded every 5 °C over a 20–95 °C temperature range with 5 min of equilibration time for each temperature. The reversibility of changes was monitored by recording the spectrum at 20 °C after cooling the samples from 95 °C in 20 min.

Gold nanoparticles were analyzed by transmission electronic microscopy, differential centrifuge sedimentation and DLS in batch mode, which showed the presence of monodispersed gold nanoparticles with a diameter of 14 nm (supplementary material, Table 1 and SI 1–2).23 

Human serum albumin and gold nanoparticles were mixed and the complexes purified by AF4 to remove free HSA and free AuNPs. HSA bound to AuNP and free HSA were analyzed separately. The SRCD was used to irradiate the sample, with a high photon flux between 185 and 260 nm corresponding to UV-C, to monitor change in the secondary structure of free HSA and HSA bound to AuNPs (ratio 1:1000). Unbound HSA was removed from HSA-AuNP complexes by field flow fractionation as previously described17 (Fig. SI-3).

The gold nanoparticles have strong UV-Vis absorbance in the 200 nm region and the classical, size-dependent, localized plasmon resonance band at around 520 nm. Citrate-stabilized AuNPs in solution do not give CD signal in the UV-Vis region, and in any case the reported CD spectra are baseline corrected with the CD spectra obtained with buffer only (for free HSA) or the CD spectrum of AuNPs only at the same concentration than in AuNP-HSA complexes. AuNPs can cause problems in CD spectra of AuNP-protein complexes. Indeed, they do not contribute to the CD signal, but they can strongly reduce the transmitted light. One of the major advantages of using a high-photon source such the synchrotron B23 beamline at Diamond Light Source is its ability to detect CD spectra of NP-protein complexes using a 10-cm pathlength.

The SRCD spectra of a solution of HSA at 2 μg/ml collected as 30 consecutive scans (equivalent to 90 min of UV exposure) in the 10 cm long cell showed that, after 30 consecutive scans, the high-photon flux from the synchrotron source induced a significant decrease in the α-helical content of free HSA as shown by changes in the amplitude of the double negative peak at 222 and 209 nm and the positive peak at 191 nm [Fig. 1(a)]. In contrast, there was no significant change over time in the secondary structure of HSA adsorbed onto AuNPs [Fig. 1(b)]. This suggests that the adsorption of HSA onto AuNPs protected it from the partial denaturation induced by UV radiation. UV-induced decrease in the helical content of free HSA (followed by monitoring the peak intensity at 209 nm) increased as a function of time, whereas the α-helical content of HSA-AUNP complexes remained constant [Fig. 1(c)].The data for free HSA were fitted to a single exponential function in agreement with a first order kinetics and a calculated rate constant of 0.030 ± 0.001 min−1, whereas the data collected for HSA bound to AuNPs were fitted to a linear function [Fig. 1(c)].

Fig. 1.

(a) Selected SRCD spectra of free HSA (2 μg/ml, cell path-length 10 cm). (b) Selected SRCD spectra of purified AuNP-HSA complex. (c) Intensity of the SRCD signal at 209 nm as a function of UV-exposure time for free HSA (red circles) and for HSA bound to AuNP (black squares). The data collected for free HSA were fitted to a single exponential function (blue line). The data collected for HSA bound to AuNPs were fitted to a linear function (orange line).

Fig. 1.

(a) Selected SRCD spectra of free HSA (2 μg/ml, cell path-length 10 cm). (b) Selected SRCD spectra of purified AuNP-HSA complex. (c) Intensity of the SRCD signal at 209 nm as a function of UV-exposure time for free HSA (red circles) and for HSA bound to AuNP (black squares). The data collected for free HSA were fitted to a single exponential function (blue line). The data collected for HSA bound to AuNPs were fitted to a linear function (orange line).

Close modal

The binding of HSA to AuNP is a complex process that is difficult to predict a priori. One of the key parameters in the NP–protein interactions is the surface charge of the NPs (Ref. 18) and its interplay with the surface charges on the proteins. Citrate-stabilized AuNPs are negatively charged at pH 7.4 and HSA, having an isoelectric point of 4.7, is also overall negatively charged at that pH. The formation of a stable complex of AuNPs with a layer of HSA on top points to a situation where HSA probably replaces the citrate molecules and acts as a stabilizer for the colloid system.

To better understand the damage induced in HSA by UV exposure, we calculated the changes in secondary structure for each one of the 30 SRCD spectra collected for free HSA and AuNP-HSA samples using the integrated software cdapps.19 UV radiation caused a 30% decrease in α-helical structure of free HSA, and a slight increase of ∼10% in β-strand and unordered structures (Fig. 2). In contrast, the secondary structure of HSA bound to AuNP showed minimal changes during UV irradiation (Fig. SI-4). AuNP could protect the proteins from UV-induced damage by absorbing part of the UV radiation, by scavenging the free radicals generated by the intense radiation1 or by both mechanisms.

Fig. 2.

UV-induced changes in the secondary structures of free HSA alone as a function of time.

Fig. 2.

UV-induced changes in the secondary structures of free HSA alone as a function of time.

Close modal

The stabilizing effect of AuNPs could also be demonstrated by monitoring the thermal unfolding of both free and bound HSA. Thermal unfolding was assessed by recording the intensity of the CD signal at 222 nm (characteristic of the α-helical structure) as a function of the temperature (Fig. 3).

Fig. 3.

Thermal unfolding (cell path-length 1 cm) of free HSA (red circles) and AuNP-HSA complexes (blue circles). The intensity of CD at 222 nm was recorded as a function of the temperature from 20 to 95 °C. Experimental data were fitted to a two-state unfolding equation (solid lines).

Fig. 3.

Thermal unfolding (cell path-length 1 cm) of free HSA (red circles) and AuNP-HSA complexes (blue circles). The intensity of CD at 222 nm was recorded as a function of the temperature from 20 to 95 °C. Experimental data were fitted to a two-state unfolding equation (solid lines).

Close modal

The fitting of the experimental data to a Boltzmann type equation A+(B − A)/(1 + exp(x − x0)/dx) (where x is the temperature, x0 the melting temperature, and dx the width of the thermal transition) using a nonlinear square fitting algorithm with the software program origin (OriginLab) gave a melting temperature of 69 °C for free HSA and of 75.4 °C for the AuNP-HSA complexes. This suggests that AuNPs increase the thermal stability of bound HSA. However, the thermal unfolding of the AuNP-HSA complexes was not completely reversible and the decrease in CD signal could result from a decrease in α-helical structure and in protein aggregation, suggesting that AuNPs likely stabilize HSA both by increasing its thermal stability and by reducing its propensity to aggregate in agreement with the fact that AuNPs suppress the aggregation of insulin and alcohol dehydrogenase.14 It would be interesting to see if gold nanoparticles could also help in stabilizing clinically important cargo molecules, such as microribonucleic acids20 by shielding them from enzymatic degradation.

This study shows that AuNPs increase the melting temperature of HSA. In a previous study, we have shown that silver nanoparticles decrease the thermal stability of HSA, while gold nanoparticles did not significantly change it.4 However, in the previous study, free HSA was not removed from the AuNP-HSA complexes, whereas the results reported here were obtained on AuNP-HSA complexes purified by AF4 fractionation, which do not contain free HSA.

The stability of human serum albumin is lower at low protein concentration, in the absence of fatty acids,21 and at low ionic strength. In particular, HSA is more stable (+0.4 kcal/mol) in the presence of 500 mM NaCl compared to 10 mM NaCl.22 Under these suboptimal conditions, HSA is quite unstable if kept at room temperature for an extended period of time. CD spectra of free HSA and AuNPs-HSA stored at 4 and at 22 °C were recorded every day for 1 week. While there was almost no change in the secondary structure of free HSA kept at 4 °C [Fig. 4(a)], the protein kept at room temperature showed a large change in the CD signal and a strong decrease in the α-helical content [Fig. 4(b)]. In contrast, there was no significant change in α-helical content of HSA in the presence of AuNPs over one week either at 4 °C [Fig. 4(c)] or at 22 °C [Fig. 4(d)]. The experiment was performed with several AuNP/HSA ratios. Free HSA lost about 80% of its α-helical structure after one week at room temperature, whereas the presence of AuNP at 1:400 and 1:5000 NP to protein ratios almost completely prevents the loss of HSA α-helical structure. HSA retained almost 80% of its α-helical content after a week at HSA/AuNP 1:10 000 ratio, suggesting that AuNPs have a strong protective effect toward the denaturation and/or the aggregation of HSA kept at suboptimal conditions.

Fig. 4.

Circular dichroism spectra of HSA (5 μg ml−1,cell path-length 1 cm) stored for one week at 4 and 22 °C in absence or presence of AuNPs. HSA at days 0 and 7 at 4 °C (a) and 22 °C (b). HSA + AuNPs (AuNPs/HSA ratio 1:400) at days 0 and 7 at 4 °C (c) and 22 °C (d). Variation of α-helical content in free HSA and AuNP-HSA at 4 °C (e) and 22 °C (f) as a function of time.

Fig. 4.

Circular dichroism spectra of HSA (5 μg ml−1,cell path-length 1 cm) stored for one week at 4 and 22 °C in absence or presence of AuNPs. HSA at days 0 and 7 at 4 °C (a) and 22 °C (b). HSA + AuNPs (AuNPs/HSA ratio 1:400) at days 0 and 7 at 4 °C (c) and 22 °C (d). Variation of α-helical content in free HSA and AuNP-HSA at 4 °C (e) and 22 °C (f) as a function of time.

Close modal

Gold nanoparticles stabilize human serum albumin exposed to “stressing” conditions, such as UV radiation, high temperature, and storage in suboptimal conditions. HSA molecules adsorbed onto AuNPs are protected from the decrease in α-helical content induced by UV irradiation and low ionic strength buffers at room temperature. In addition, HSA complexed to AuNPs has a higher melting temperature than free HSA, which strengthens the fact that AuNPs stabilize HSA secondary structure.

This stabilizing effect could be useful in biotechnology, where AuNPs are used as biocompatible stabilizing agents for proteins in deleterious conditions.

The authors would like to thank Diamond Light Source for beamtime on B23 beamline (SM9836 and SM9064), Támas Jávorfi of B23 beamline and Sylvain D. Vallet (ICBMS, Lyon, France) for technical assistance. This work has been carried out within the JRC Institutional research action 15524 (Nanobiosciences). The authors thank to European Commission for funding.

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Supplementary Material