Ajith Pattammattelabc,
Christina L. Williamsab,
Paritosh Pandea,
William G. Tsuia,
Ashis K. Basua and
Challa Vijaya Kumar*abc
aDepartment of Chemistry, University of Connecticut, 55 North Eagleville Road, Unit 3060, Storrs, CT 06269-3060, USA. E-mail: Challa.Kumar@UCONN.edu
bDepartment of Molecular and Cell Biology, University of Connecticut, 55 North Eagleville Road, Unit 3060, Storrs, CT 06269-3060, USA
cThe Institute of Material Science, University of Connecticut, 55 North Eagleville Road, Unit 3060, Storrs, CT 06269-3060, USA
First published on 1st July 2015
The influence of oxidative debris (OD) present in as-prepared graphene oxide (GO) suspensions on proteins and its toxicity to human embryonic kidney cells (HEK-293T) are reported here. The OD was removed by repeated washing with aqueous ammonia to produce the corresponding base-washed GO (bwGO). The loading (w/w) of bovine serum albumin (BSA) was increased by 85% after base washing, whereas the loading of hemoglobin (Hb) and lysozyme (Lyz), respectively, was decreased by 160% and 100%. The secondary structures of 13 different proteins bound to bwGO were compared with the corresponding proteins bound to GO using the UV circular dichroism spectroscopy. There was a consistent loss of protein secondary structure with bwGO when compared with proteins bound to GO, but no correlation between either the isoelectric point or hydrophobicity of the protein and the extent of structure loss was observed. All enzymes bound to bwGO and GO indicated significant activities, and a strong correlation between the enzymatic activity and the extent of structure retention was noted, regardless of the presence or absence of OD. At low loadings (<100 μg mL−1) both GO and bwGO showed excellent cell viability but substantial cytotoxicity (∼40% cell death) was observed at high loadings (>100 μg mL−1). In control studies, OD by itself did not alter the growth rate even after a 48 h incubation. Thus, the presence of OD in GO played a very important role in controlling the chemical and biological nature of the protein–GO interface and the presence of OD in GO improved its biological compatibility when compared to bwGO.
Structural denaturation of proteins at GO, because of unfavorable interactions between GO and the hydrophobic protein interior, adversely affects the protein function.4 Thus, several surface passivation approaches were established to mask unfavorable hydrophobic interactions5 to prevent protein denaturation. Modulation in enzyme properties such as, decrease or increase in enzymatic activity,4–6 and complete inhibition7 on binding of enzymes to GO was illustrated before. The conformation and protein structure as well as its orientation at the nanosurface play major roles in determining bound enzyme activities.7
Chemical functionalization,8 reduction, and passivation with intermediary proteins9,10 or polymers,11 can successfully passify GO surface and stabilize certain proteins and enzymes. Reports suggest that the extent of surface hydrophobicity plays a major role in retaining protein structure and thereby bound enzyme function.5 Recent advances in structural studies of GO have identified the presence of small, highly oxidized polycyclic aromatic moieties called oxidative debris (OD) in GO suspensions.12 In addition, decrease in conductivity,13 increase in fluorescence,16 increased electrochemical activity14 and decreased interactions of GO with small molecules15 are attributed to the presence of OD on its surface. Treating GO with aqueous base solutions separates this debris (Scheme 1), and the resulting base-washed GO (bwGO) has several desirable and improved properties.16 Thus, an interesting question arises as to how and to what extent OD disturbs biological properties of GO? To date, no such investigation has been carried out to investigate the role of OD in controlling the interactions of GO with biological molecules or cells. Biological applications of GO are being currently actively pursued for a variety of reasons.1,17 Therefore, it is critical to analyze the nature of bio–GO interface in the absence of OD. Moreover, the structure of bwGO is closer to graphene than to GO and, therefore, it is important to examine the influence OD present in GO on its interactions with proteins, enzymes and other biomolecules.
Here, we report the role of OD at GO interface in controlling the properties of a set of 13 different proteins. These have increasing isoelectric points (pH where the net charge on the protein is zero, pI), molecular weights, and increasing number of surface arginines (ESI, Table S1†). Our results suggest that OD plays a major role in controlling binding affinities, as well as bound enzyme structure/activities. Binding to GO and bwGO, structure retention and enzymatic activities were analyzed using multiple methods (Scheme 1). Furthermore, cytotoxicity of GO, bwGO as well as OD and differences in their toxicities are examined here. Our current study gives an insight into the fundamental understanding of bio–GO interactions at molecular level such as the role of surface functionalities and their nature in determining the affinity, secondary structure, and enzymatic activities. This information would be valuable for the rational control of protein behavior at particular nanosurfaces.
![]() | (1) |
Q = KCeQsat/[1 + KCe] | (2) |
Briefly, 0.5 × 105 cells were seeded in each well of a 24 well plate in 500 μL of complete growth media [Dulbecco's Modified Eagle media (DMEM) supplemented with 10% fetal bovine serum (FBS)] and incubated for 24 h to achieve a metabolically active early-log phase (1.0 × 105 cells). The GO, bwGO and OD samples were dispersed in phosphate buffered saline (PBS, 10 mM, pH 7.0) and sonicated for 1 h as reported earlier.25
The suspensions were further diluted with cell culture media and quickly vortexed before they were introduced to the adherent cells at a concentration range of 10, 25, 50, 75, 100, 250 and 500 μg mL−1. During the co-incubation phase, the cell morphology was constantly monitored using light microscopy. The intracellular metabolic rate was assessed after 24 h of co-incubation by using CCK-8 kit that contains a tetrazolium salt, WST-8, which got reduced by dehydrogenase activity of live cells to generate a yellow-colored formazan dye. The amount of formazan dye generated, was quantified spectrophotometrically at 450 nm and it is directly proportional to the number of living cells. Appropriate negative controls, where the CCK-8 kit reagent (50 μL per well) was substituted with equal volume of PBS, were used for each concentration of GO, bwGO and OD whereas the positive control consisted of 50 μL of WST-8 solution added to the pristine cells grown in 500 μL of growth media. The data were analysed by standard statistical methods.
Eqn (1) was used to fit the quenching data where [Q] = quencher concentration (GO/bwGO); F0 = fluorescence intensity in the absence of quencher; F = fluorescence intensity at given [Q]; Ka = bimolecular affinity constant, and fa = fraction of the initial fluorescence. Thus, from the slope (1/Kafa) and y-intercept (1/fa) of the fitted line, Ka was calculated.
The quenching constants were compared with the assumption that both bwGO and GO quench the fluorescence to the same extent, and Ka for GO 5.7 (±0.8) × 103 mL mg−1 and 6.8 (±1.2) × 103 mL mg−1. Clearly, the affinity of BSA increased when OD has been removed.
The binding of proteins to GO and bwGO was also investigated in equilibrium binding studies, where the samples were equilibrated with protein solutions, and unbound protein has been separated by centrifugation. Unbound protein concentration was determined by its absorbance at 280 nm for BSA and lysozyme, while absorbance at 406 nm has been monitored for Hb samples. The corresponding adsorption isotherms are shown in Fig. 2.
The affinity of BSA increased after the base wash (Fig. 2A, green), which is in good agreement with the above fluorescence studies. In the cases of both Hb and Lyz, the binding to bwGO was similar to that of GO (Fig. 2B and C, red lines for GO and green lines for bwGO) at low protein concentrations of 5–10 μM for Hb and 10–20 μM for Lyz. But at higher concentrations (>10 and >20 for Hb and Lyz, respectively), protein loading on bwGO was less than that of GO, under the same conditions. Maximum loading (w/w) of BSA was increased after base-wash by 85%, that of Hb decreased by 160% and that of Lyz decreased by 100%. The binding of GOx to GO was negligible at low protein concentrations, while bwGO showed a maximal loading of 64% (ESI Fig. S3†), and in case of GO it decreased to 52%.
Langmuir model of Hb and Lyz adsorption to the nanosheets showed clear differences in binding affinities after base wash (Table 1). The Ka for Hb to GO was 1.9 (±0.6) × 107 M−1, whereas the affinity decreased substantially for bwGO (3.6 (±2.1) × 106 M−1). Lyz showed strong adsorption to GO with Ka of 8.1 (±3.5) × 107 M−1, and it decreased to 2.8 (±0.8) × 107 M−1 for bwGO. The decrease in affinity is also reflected in the adsorption parameter, Qsat, which represents theoretical maximum for monolayer formation of the protein (in μmol) per solid (in mg). As expected, Hb and Lyz showed significant drop in maximal loadings (2–3 fold), which suggests weaker adsorption of proteins to bwGO.
System | Ka (M−1) | Maximal loading, Qsat (μmol mg−1) | R2 |
---|---|---|---|
Hb/GO | 1.9 (±0.6) × 107 | 124 (±26) | 0.99 |
Hb/bwGO | 3.6 (±2.1) × 106 | 38 (±8) | 0.94 |
Lyz/GO | 8.1 (±3.5) × 107 | 530 (±152) | 0.98 |
Lyz/bwGO | 2.8 (±0.8) × 107 | 219 (±26) | 0.97 |
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Fig. 3 Zeta potential titrations of Hb (A), and Lyz (B) showed gradual charge neutralization during protein adsorption to GO (red) and bwGO (green). |
The ratio of ellipticities at 222 nm of GO-bound protein to that of the unbound protein (RE@222) indicated the order GOx (pI – 4.6) > RNase (9.3) ≥ trypsin (9.3) ≥ ovalbumin (4.9) = catalase (5.4) > Lyz (11.3) > pepsin A (1.0) ≥ BSA (5.5) ≥ BLG (5.1) > Cyt c (10) > Hb (6.8) > HAS (4.7) > Mb (6.8) (Fig. 4). In the case of bwGO, the trend was trypsin > GOx > RNase ≥ BLG ≥ pepsin A > Lyz ≥ ovalbumin = catalase > BSA > Hb ≥ Cyt c > HSA > Mb, but in almost all cases, the extent of structure retention was lower with bwGO than with GO.
A plot of the relative loss of structure when the protein is bound to GO vs. bwGO was generated (Fig. 4B) where the relative loss (ΔRE@222) is defined as RE@222 of a protein bound to bwGO minus the RE@222 of the same protein bound to GO. A positive value of this parameter indicates gain in protein secondary structure while a negative value corresponds to further loss in structure due to base-wash. The data show that maximal loss in structure occurred when the pI of the protein is close to neutral value, or when the protein has ∼−15 charge. This consistent loss in secondary structure could result in decreased enzymatic activities, and hence activities of enzymes bound to bwGO were determined and compared with those bound to GO.
Oxidase activity of GO/GOx was essentially the same (94%) as that of the pristine GOx, but significant reduction has been noted (65%) in case of bwGO/GOx. Peroxidase-like activity of Hb was only 20% upon binding to GO and it reduced further to 5% on binding to bwGO. Similarly, GO/Mb showed only 14% activity and no measurable activity has been noted for bwGO/Mb. In case of Cat, the activity was reduced to ∼60% on binding to GO while the activity of bwGO/Cat has been decreased to 45%.
A plot of loss in activity vs. enzyme charge (Fig. 5B) indicated a poor correlation with charge, which suggests that electrostatic interactions do not control the enzyme activity at this interface. To further test these conclusions, we have examined the influence of these materials on cell growth or cell viability using HEK 293T cells.
The above intracellular metabolism results were substantiated by light microscopy images taken at 24 h and 36 h of co-incubation, which revealed significantly stressed cell morphology at the GO and bwGO concentrations beyond 75 μg mL−1. Interestingly, at greater than 75 μg mL−1, bwGO turned out to be slightly more cytotoxic than GO.
We also examined the cells exposed to OD isolated from washing GO, but no morphological or metabolic differences could be observed between cells incubated with OD and control HEK 293T cells. The results obtained from CCK-8 kit, for monitoring intracellular dehydrogenase activity and light microscopy for assessing the cell morphology correlated consistently throughout the trials.
To further investigate the influence of OD on these cells, they were detached and transferred to a six-well plate and monitored for an additional 48 h (this is beyond the first 36 h of co-incubation). By this time cells exposed to the higher doses of GO and bwGO started to die, whereas the control cells and cells exposed to OD were still metabolically active. No appreciable difference in the rate of cell division, morphology or cellular metabolism was observed between OD co-incubated and control cells.
In summary, the binding affinities of several proteins have decreased, and their structure retention and enzymatic activities (when relevant) have also been decreased when the OD has been removed from the GO suspensions. Thus, OD appears to play an important role in shielding these bio-macromolecules from any adverse interactions of the underlying graphitic surface. When the surface is coated with low loadings of BSA (400% (w/w)), the activities of both Hb and Mb have recovered and even exceeded those adsorbed onto GO. Cytotoxicity studies show that these materials are toxic to HEK 293T cells at high concentrations (>75 μg mL−1) and long exposure times (>24 h).
Adsorption of proteins to both GO and bwGO are marginally different when evaluated against a small set of 13 proteins whose pI values ranged from 4 to 12. The maximum loading observed for Hb (pI 6.8) with GO was 320% (w/w), and this translates into an average of ∼1.3 layers of Hb on the nanosheets, if we assume that the protein occupies the entire surface (7.05 × 10−22 Å2 g−1) and that the protein diameter is unchanged upon binding to the nanosheets. Along these lines, Hb binding to bwGO saturated around an average of 0.7 layers, much less than that observed with GO. This decrease in the coverage could be due to at least two possible factors, (1) decrease in intrinsic affinity of Hb to bwGO, or (2) loss in the secondary structure of bound Hb such that it occupies a larger area on the nanosheets. In support of the former, the binding affinity of Hb decreased 10-fold, from GO to bwGO. On the other hand, the CD data analysis indicated only 10% loss in the CD band intensities for Hb bound to bwGO when compared to that bound to GO. Therefore, the decrease in the maximum loading of Hb is more likely due to reduced affinity.
Similar analysis of the CD data of Lyz (ΔRE@222 = −18%), GOx (ΔRE@222 = −14%) and BSA (ΔRE@222 = −23%) also indicates that protein denaturation is not directly controlling the loading maxima. Therefore, the changes in interactions at bwGO vs. GO could be responsible for the differences in affinities.
The function of biohybrids can be quite sensitive to the conformation of the bound protein.30,31 Current studies involving 13 different proteins revealed that there has been a small but consistent increased loss in protein structure with bwGO when compared to the proteins bound to GO. To further understand the basis for increased protein structure loss on bwGO, we examined if there is any correlation between structure loss and protein charge or the hydropathy index of the protein. The average hydropathy index32 was calculated using Expasy Protparam tool and it indicated the order Cyt c > RNase A > catalase > BSA = Lyz > HSA = Mb > GOx > BLG > trypsin > ovalbumin > Hb > pepsin A, but this trend has no correlation with the observed trends in RE@222 or ΔRE@222 of these proteins bound to bwGO.
The relative loss of ellipticity at 222 nm (ΔRE@222), which is a measure of the per cent structure retention when compared to that of the unbound protein, did not correlate with net charge on the protein (ESI, Fig. S6†), or the lysine content of the protein (ESI, Fig. S7†) or the sum of the number of lysine and arginine residues present in the protein (ESI, Fig. S8†).
On the other hand, the differences in the extents of structure loss when proteins bind to bwGO vs. GO (ΔRE@222), however, depended on the percentage arginine content of the protein as well as the aliphatic index of the protein (Fig. 7B). Twelve proteins containing arg contents of 0–7% showed strong correlation with the extent of structure loss (Fig. 7A), irrespective of their net charge. This strong trend shows the critical role of Arg residues in the interactions with bwGO. Arg was suggested to interact strongly with GO because of its ability to form hydrogen bonds as well as its charge and hydration status.33–35 Lyz with 8% Arg content deviated significantly from the plot and retained greater extent of structure due to its unusually high thermodynamic stability.36 In a recent study, strong interaction of lysozyme, an arg rich protein, with carbon nanotubes (CNT) was demonstrated,37 and the binding free energy (ΔGbind) between strongly interacting Args in Lyz with CNT was −5.9 kcal mol−1, higher than that of lysine (−3.5 kcal mol−1).38
Interaction of the nanosurface with the amino acid side chains after base wash would influence bound protein conformation.5 The possible role of hydrophobic residues in distorting the structure of the bound protein (between bwGO and GO) is examined in Fig. 7B. The plot of extent of relative structure loss (ΔRE@222) as a function of the average aliphatic index showed a strong correlation. Aliphatic index is the volume occupied by the side chains of aliphatic amino acids (alanine, valine, isoleucine, and leucine) of the protein. There has been a greater retention of protein secondary structure with increasing aliphatic index. Base washing had less and less influence as the aliphatic index increased. That is, more hydrophobic proteins did not distinguish between bwGO and GO while less hydrophobic proteins are more sensitive to exposure to the hydrophobic surfaces of bwGO. Thus, the role of OD in these interactions depends also on the aliphatic index of the protein. Therefore, these afore mentioned correlations show that the interaction of bwGO with biomolecules is primarily via charged arginine as well as hydrophobic side chains, along with other specific interactions with surface functional groups of the nanosolid.
Correlation of the enzymatic activities of the enzymes bound to GO and bwGO was another tool used to compare the effect of OD. In support of our secondary structure studies, most showed decreases in activities when bound to bwGO vs. GO, Fig. 5A. Peroxidase like activity of Hb and Mb, reductase activity of Cat and oxidase activity of GOx decreased at both interfaces. There has been no correlation with enzyme charge. Thus, hydrophobic interactions discussed above could be responsible for the structure loss which could result in activity loss. This possibility was examined next.
Further insight into the protein–GO interactions, thus, was evident when relative activities of the bound proteins are compared with their corresponding extents of secondary structure retention. A good linear correlation between enzyme secondary structure and enzymatic activity has been noted for Hb, Mb, Cat and GOx (Fig. 8). Evidently, structural denaturation is the primary reason for the decrease in activity of the bound enzymes as the debris has been removed. This might seem trivial as structure retention is essential for activities but it has been noted that GO inhibited the activity of chymotrypsin39 whereas GO has increased the activities of oxalate oxidase, esterase5 and cytochrome c.10
Since loss in activity is highly undesirable, we tested if the hydrophobic surfaces of bwGO could be passified by the adsorption of cationized BSA onto the nanosolid prior to enzyme loading.10 BSA was chemically modified with the polyamine, tetraethelenepentamine (TEPA), which resulted in BSA charge reversal from −20 to +23 as confirmed by agarose gel electrophoresis. The bwGO surface (0.2 mg mL−1) was first passivated with cationized BSA (400% w/w), and Hb (8 μM), Mb (12 μM), Cat (0.8 μM) or GOx (4 μM) were loaded onto the nanosolid. Activities of the above enzymes bound to cationized BSA-loaded bwGO (BSA–bwGO) are compared with those bound to bwGO, under similar conditions (Fig. 8B). Surprisingly, the activities of GOx, Hb and Mb bound to BSA–bwGO exhibited substantial improvements (Fig. 8B, blue bars) while Cat showed minor improvements. Thus, the novel biofunctionalization strategy with cationized BSA to modify high energy nanosolids10 can be successfully applied to passify bwGO for favourable enzyme loading.
Finally, the cell survival studies show that OD affects the interaction of GO with 293T cells. Incubation for 24 h, both GO and bwGO showed appreciable cytotoxicity above 75 μg mL−1 concentration (Fig. 6). The dose dependent cytotoxicity of GO beyond 75 μg mL−1 concentration is in agreement with the previously published results.23 However, to the best of our knowledge, there is no study that reported the biocompatibility of bwGO with human cell lines. Chemically reduced GO was found to be much more toxic, in comparison to GO or bwGO.40 There has been no detectable toxicity for OD, even at very high doses (500 μg mL−1), which indicates that any toxic effect of GO is intrinsic to it and not necessarily due to the presence of OD in GO, but further studies may be needed to validate this interesting observation.
Footnote |
† Electronic supplementary information (ESI) available: Characterization of GO and bwGO, data analysis and enzymatic activity assay plots. See DOI: 10.1039/c5ra10306a |
This journal is © The Royal Society of Chemistry 2015 |