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The effects of allantoin, arginine and NaCl on thermal melting and aggregation of ribonuclease, bovine serum albumin and lysozyme Tsutomu Arakawa ∗ , Nasib Karl Maluf Alliance Protein Labs, A Division of KBI Biopharma, United States
a r t i c l e
i n f o
Article history: Received 28 July 2017 Received in revised form 4 October 2017 Accepted 6 October 2017 Available online xxx Keywords: Allantoin Arginine Thermal aggregation Thermal stability Circular dichroism
a b s t r a c t Allantoin is widely used as a skin care agent and readily forms crystals, which were recently shown to bind endotoxins and high molecular weight aggregates in cell culture harvests. Here, we have investigated the effects of allantoin on thermal stability and aggregation of protein using ribonuclease, bovine serum albumin and lysozyme using temperature-regulated circular dichroism (CD) and differential scanning microcalorimetry (DSC). Ribonuclease showed no change in thermal stability and aggregation by the addition of allantoin. While allantoin showed no effects on the thermal stability of bovine serum albumin, it enhanced aggregation. Similarly, allantoin showed no stabilizing effects on lysozyme, but it strongly suppressed aggregation. Such suppressed aggregation resulted in reversibility of thermal unfolding of lysozyme. These effects of allantoin were then compared with those of NaCl and arginine hydrochloride. Arginine was similar to allantoin at low concentrations, where both solvent additives can be compared due to limited solubility of allantoin. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Allantoin, an inexpensive natural product, is a cell metabolite present in a variety of plants, including wheat germs, tobacco seeds and comfrey roots [1,2]. Allantoin is also found in animals and can be chemically synthesized from urea [3]. Allantoin has antiinflammatory, anti-ulcer and cell-growth promoting activities and is thus widely used in a variety of commercial non-pharmaceutical (e.g. cosmetic), and pharmaceutical (e.g. as a wound healing skin care agent) [4–9]. It is slightly soluble in aqueous solution and readily forms crystals, and is chemically unstable in alkaline solution. Recently, a novel application of crystalline allantoin has been reported by Gagnon et al. [10–15]. Allantoin crystals were found to bind endotoxins and high molecular weight aggregates, which contain variable amounts of chromatin, in cell culture harvests with high specificity and affinity. To our knowledge, no studies have been reported on the effects of soluble allantoin on protein solution. Allantoin is chiral as shown in Fig. 1 (circle) and contains amide groups and as such, its structure is similar to amine-containing low molecular weight compounds that have been shown to suppress protein aggregation during thermal melting [16–18]. Arginine has
∗ Corresponding author at: Alliance Protein Laboratories, 6042 Cornerstone Court West A, San Diego, CA, 92121, United States. E-mail address: [email protected] (T. Arakawa).
also been found to be a highly effective aggregation suppressor at high concentration. Compared with the synthetic amine compounds, arginine is a natural amino acid and thus safe to be used as an aggregation suppressing agent [19–23]. In this paper, we have investigated the effects of soluble allantoin on heat-induced structure changes and resultant aggregation of globular proteins and compared the results with the effects of NaCl and arginine.
2. Materials and methods Three commercial proteins, BSA (bovine serum albumin), Lyso (lysozyme) and RNase (ribonuclease), were used as received. These proteins were dissolved in phosphate-buffered saline (PBS, 10 mM phosphate, 0.15 M NaCl, pH 7.0) at 20 mg/ml and used as a stock solution. A stock solution of allantoin was prepared at 200 mM in PBS by heating at 95 ◦ C. Once dissolved, the stock solution was kept at 75 ◦ C without precipitation. The stock solution was prepared daily. Stock solution containing 1 M arginine or 2 M NaCl were also prepared in PBS. Circular dichroism (CD) analysis was carried out on a Jasco J-1500 spectropolarimeter equipped with a Peltier temperature controller. Since arginine and allantoin have strong absorbance in the far UV region, the near UV region was used for CD measurements. Near UV CD spectra and melting curves were determined using 1 cm path-length quartz cell with 1 cm width. A 2 mL aliquot
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66 65
NaCl
64 Fig. 1. Structure of allantoin.
Asymmetric carbon is circled.
63
Arginine
62 61 0
500
1000
1500
Fig. 3. Melting temperature of RNase as a function of allantoin (square), arginine (diamond) and NaCl (triangle) concentration. Melting of 4 mg/ml BSA was followed at 270 nm.
Fig. 2. Thermal melting of RNase in the absence (curve 1) and presence of 0.9 M arginine (curve 2) and 1.8 M NaCl (curve 3). Melting of 2 mg/ml RNase was followed at 276 nm.
of the samples was loaded in the cell. Melting was followed at the indicated wavelength with a temperature increment of 1 ◦ C/min. The sample and reference cells (made of tantalum) were loaded with ∼0.5 mL of degassed sample and buffer, respectively. A MicroCal VP-DSC was programmed to scan from 10 to 95 ◦ C at a rate of 60 ◦ C/hr with an 8 s filtering period and no feedback. Several buffer vs. buffer scans were recorded before running the sample vs. buffer experiment to bring the thermal environment of the instrument to a steady state and to obtain a pre-experiment buffer-buffer baseline. After each sample-buffer scan, the cell was cleaned in cycle with 10% Contrad, followed by an exhaustive water wash. The raw data were processed and analyzed using Origin 7.0. A buffer-buffer scan was subtracted from each sample-buffer scan, and the baseline was calculated and processed using the Origin software according to the manufacture’s instructions. The baseline-corrected Cp profiles were normalized to protein concentration (expressed as kcal/mol/◦ C). 3. Results and discussion 3.1. Ribonuclease The near UV CD spectra of 1.5 mg/ml RNase in PBS were compared at 20 and 90 ◦ C (data not shown), from which the optimal protein concentration and wavelength to monitor thermal melting were determined to be 2 mg/ml and 276 nm. Fig. 2 shows the change in CD intensity at 276 nm of 2 mg/ml RNase in PBS with increasing temperature from 40 to 80 ◦ C (curve 1). The negative CD signal at this wavelength started increasing at 57 ◦ C with a transition mid-temperature (Tm) of ∼64 ◦ C. This melting was accompanied by no increase in HT[V] signal at 276 nm (data not shown), which closely follows the absorbance property and hence the light scattering of the sample. It thus does not appear that ther-
mal unfolding of RNase accompanies aggregation. Fig. 2 also shows the melting curves of RNase in 0.9 M arginine (curve 2) and 1.8 NaCl (curve 3). The melting curve was shifted to a higher temperature in NaCl and to a lower temperature in arginine, indicating stabilization by NaCl and destabilization by arginine of RNase structure. Neither case involved increase in HT[V] intensity, indicating that neither NaCl nor arginine caused visible aggregation of RNase, as in the buffer solution. Fig. 3 plots the melting temperature of RNase as a function of additive concentration. It is evident that both arginine and NaCl had no effects on melting temperature at low concentrations and that at higher concentration NaCl increased the melting temperature while arginine decreased it. Allantion showed no effects on melting temperature and HT[V] signals of RNase, although higher concentration of allantoin could not be used due to its limited solubility. It may be summarized here that RNase does not appear to aggregate upon melting and that NaCl and arginine have an opposite effect on its thermal stability. Allantoin is inert to thermal stability and aggregation of RNase. 3.2. BSA Similarly, the near UV CD spectra of 2 mg/ml BSA in PBS were compared at 30 and 90 ◦ C (data not shown), from which the optimal wavelength to monitor BSA melting was determined to be 270 nm. Fig. 4A shows the melting of 4 mg/ml BSA in PBS (curve 1). The CD intensity at 270 nm started increasing at ∼55 ◦ C with a Tm of 65 ◦ C. In this case, the HT[V] intensity started increasing around 55 ◦ C as shown in Fig. 4B (curve 1), reflecting thermal unfolding of BSA. Fig. 4A also shows the melting curves of 4 mg/ml BSA in 0.8 M arginine (curve 2) and 0.8 M NaCl (curve 3). Both additives shifted the melting curves to higher temperature, stabilizing the BSA. Fig. 5 plots the Tm against arginine (diamonds) and NaCl (triangles) concentration. While NaCl linearly increased the Tm, arginine appeared to level-off at 500 mM. Fig. 5 also shows the effects of allantoin (squares), which resulted in no changes in Tm up to 100 mM. BSA at 4 mg/ml showed aggregation above 55 ◦ C as described above (Fig. 4B). Fig. 4B also shows the HT[V] signal at 270 nm as a function of temperature in 10 mM (curve 2) and 40 mM (curve 3) allantoin. It is evident that the rate of HT[V] increase was greater in the presence of allantoin, more so at 40 mM and thus that allantoin enhanced aggregation of BSA. The difference in HT[V] (HT[V]) between 40 and 80 ◦ C is plotted in Fig. 6 as a function of arginine (diamonds), NaCl (triangles) and allantoin (squares) concentration. Allantoin resulted in a steady increase in HT[V], indicating its enhancing effects on BSA aggregation. Arginine (diamond) initially followed the same trend, sharply increasing HT[V]. However, the
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Fig. 4. Thermal melting (A) and HT[V] of BSA (B). A: No additive (curve 1), 0.8 M arginine (curve 2), 0.8 M NaCl (curve 3). B: No additive (curve 1), 10 mM allantoin (curve 2), 40 mM allantoin (curve 3).
the three additives showed a different behavior on BSA. Allantoin showed no effects on melting temperature, but enhanced aggregation. Arginine increased the melting temperature slightly and enhanced aggregation at low concentration, but reduced aggregation at high concentration. NaCl showed weak enhancement of BSA aggregation, while significantly increasing the melting temperature.
69 68 67 66
3.3. Lysozyme
65 64 0
200
400
600
Fig. 5. Melting temperature of BSA as a function of allantoin (square), arginine (diamond) and NaCl (triangle) concentration.
70 60 50 40 30 20 10 0
0
200
400
600
800
Fig. 6. HT[V] of BSA as a function of allantoin (square), arginine (diamond) and NaCl (triangle) concentration.
HT[V] reached a plateau around 100 mM arginine, after which the HT[V] gradually decreased with increasing arginine concentration, eventually leading to a value smaller than in the absence of arginine. NaCl (triangles) resulted in a small increase in HT[V] both at low and high NaCl concentrations. An interesting observation is that the increase in HT[V] was smaller in NaCl than arginine at 100 mM, but greater in NaCl at 800 mM. It is thus concluded that
Comparison of the near UV CD spectra of 1 mg/ml Lyso in PBS at 25 and 90 ◦ C indicated the optimal wavelength to be 288 nm. Fig. 7A shows the melting curve of Lyso in PBS (curve 1), indicating a commencement of melting at 68 ◦ C with a Tm of 73–74 ◦ C. The melting temperatures were unchanged in the presence of allantoin, arginine and NaCl all up to 100 mM (data not shown). Namely, they do not affect the thermal stability of Lysozyme, perhaps due to its inherent high thermal stability. Fig. 7B shows the change in HT[V] at 288 nm with increasing temperature in the absence (curve 1) and presence of 5 mM (curve 2), 10 mM (curve 3) and 100 mM (curve 4) allantoin. The HT[V] signal at 288 nm started increasing around 75 ◦ C followed by a sharp increase, indicating extensive aggregation. The rate of HT[V] increase was slowed considerably with increasing allantoin concentration, as clearly seen in Fig. 8 (squares). At 100 mM, allantoin appeared to fully suppress Lyso aggregation, as HT[V] was close to zero. This was confirmed by demonstrating that Lyso unfolding under these conditions was fully reversible. Fig. 7A shows a second thermal scan (curve 2) after heating to 92 ◦ C in the first scan, indicating full reversibility by this technique. Fig. 8 also shows HT[V] in arginine (diamonds) and NaCl (triangles). There were no changes in NaCl, indicating that NaCl neither increased nor decreased thermal aggregation of Lysozyme. Arginine (diamonds) showed a similar trend to allantoin, suppressing thermal aggregation. It may be concluded that while allantoin, arginine and NaCl showed no effects on Lyso thermal stability, allantoin and arginine showed strong suppression on thermal aggregation of Lysozyme. The effect of allantoin on the thermal denaturation properties of Lyso was also studied using DSC, which can provide more detailed information on thermal unfolding. Fig. 9 shows the heat capacity profiles for the thermal denaturation of a 1 mg/ml Lysozyme solution in the absence and presence of 100 mM allantoin. Fig. 9 shows the raw heat capacity data (i.e. not baseline corrected), while
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Fig. 7. Thermal melting (A) and HT[V] (B) of Lyso. Melting of 1 mg/ml Lyso was followed at 288 nm. A: scan 1 (curve 1), scan 2 (curve 2). B: non additive (curve 1), 5 mM allantoin (curve 2), 10 mM allantoin (curve 3), 100 mM allantoin (curve 4).
lysozyme, scan 1 lysozyme + allantoin, scan 1 lysozyme + allantoin, scan 2
140 120
1
100
10
80
2
60 40 5
20 0
0
50
3
100
Fig. 8. HT[V] of Lyso as a function of allantoin (square), arginine (diamond) and NaCl (triangle) concentration.
0 55
60
65
70
75
80
85
Fig. 10. Baseline-corrected heat capacity profile. Curve 1: no additive. Curve 2: scan 1 with 100 mM allantoin. Curve 3: scan 3 with 100 mM allantoin.
Fig. 9. Heat capacity profile of Lyso. Curve B, buffer. Curve 1 (scan 1) and 2 (scan 2): no additive. Curve 3 (scan 1), 4 (scan 2), 5 (scan 3) and 6 (scan 4): 100 mM allantoin.
Fig. 10 shows baseline corrected data for selected scans. In Fig. 9, the red trace (curve 1) corresponds to the denaturation of Lyso (in the absence of allantoin), and gives a Tm of 72.84 ◦ C, which is consistent with the CD melting result. Note that the shift to lower heat capacity values from the buffer-buffer scan (curve B) to the lysozyme-buffer scan is due to the displacement of buffer by protein, since buffer (which is mostly water) has a higher heat capacity than protein. The blue trace (curve 2) corresponds to a repeat scan of the same material; after completion of the first denaturation scan, the instrument rapidly cooled the sample cell from the highest temperature of 95 ◦ C down to 10 ◦ C, held it at that temperature for 15 min, and then initiated the second thermal denaturation scan. Thus, this experiment investigated the extent to which Lyso would reversibly fold into the native structure after thermal denaturation. The blue trace (curve 2) shows that under these conditions and given these particular denaturation parameters, its thermal denaturation is completely irreversible, consistent with the observed extensive aggregation from the CD experiments (Fig. 7B, curve 1).
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When this experiment was carried out in the presence of 100 mM allantoin (the green trace, curve 3, representing the first scan), we can see that approximately 40% of the total protein was able to refold under these conditions (scan 2, curve 4, shown in magenta), and again for scan 3 (the cyan trace, curve 5) a small amount of heat was detected, indicating that a small amount of refolding occurred. The orange trace (curve 6) shows that no observable refolding was detected for scan 4. Thus, allantoin promotes the refolding of thermally denatured Lyso, consistent with the CD experiments. However, in contrast to the CD experiment, we did not observe 100% refolding, which may be due to the fact that the heating and cooling parameters between the two techniques were not identical and that thermally denatured Lyso were exposed to different cell surface (quartz cell for CD and tantalum cell for DSC). The Tm for unfolding of Lyso in the presence of 100 mM alantoin was 72.97 ◦ C and 72.61 ◦ C for scans 1 and 2, respectively (see Fig. 10), which are essentially indistinguishable from each other. However, the difference in the total unfolding enthalpy observed between the first thermal denaturation scans for Lyso in the absence and presence of allantoin is almost certainly outside the normal variability observed for this technique, and suggests that allantoin does affect the total heat flow observed during the melting process. The total unfolding enthalpies for the baseline corrected scans shown in Fig. 10 (the total area under the curve) are 90 kcal/mol, 73 kcal/mol and 27 kcal/mol for lysozyme alone, Lyso + allantoin (scan 1) and lysozyme + allantoin (scan 2), respectively. While no change in melting temperature by allantoin suggests its binding to both native and unfolded states of Lysozyme, it is not clear why the enthalpy of unfolding was lower in the presence of allantoin. 4. Conclusion Three proteins were used to examine the effects of allantoin, arginine and NaCl on thermal aggregation in PBS. First, RNase did not appear to aggregate upon melting in PBS. NaCl and arginine had opposite effects on its thermal stability. Allantoin was inert to thermal stability and aggregation of RNase. These 3 additives showed a different behavior on BSA. Allantoin showed no effects on melting temperature, but enhanced aggregation. Arginine increased the melting temperature slightly and enhanced aggregation at low concentration, but reduced aggregation at high concentration. NaCl showed weak enhancement of BSA aggregation, while significantly increasing the melting temperature. Against lysozyme, allantoin, arginine and NaCl showed no effects on thermal stability. On the contrary, allantoin and arginine showed strong suppression on thermal aggregation. Allantoin at 100 mM resulted in reversible thermal melting.
[2] [3] [4] [5] [6]
[7]
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[10]
[11]
[12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
[20] [21] [22] [23]
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´ V.D. Nikolic, ´ I.A. Arsic, ´ L.P. Stanojevic, ´ S.J. Najman, S. Stojanovic, ´ I.I. [1] V.L.J. Savic, ´ ´ Comparative study of the biological activity of Mladenovic-Ranisavljevi c,
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ARTICLE IN PRESS
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International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
The effects of allantoin, arginine and NaCl on thermal melting and aggregation of ribonuclease, bovine serum albumin and lysozyme Tsutomu Arakawa ∗ , Nasib Karl Maluf Alliance Protein Labs, A Division of KBI Biopharma, United States
a r t i c l e
i n f o
Article history: Received 28 July 2017 Received in revised form 4 October 2017 Accepted 6 October 2017 Available online xxx Keywords: Allantoin Arginine Thermal aggregation Thermal stability Circular dichroism
a b s t r a c t Allantoin is widely used as a skin care agent and readily forms crystals, which were recently shown to bind endotoxins and high molecular weight aggregates in cell culture harvests. Here, we have investigated the effects of allantoin on thermal stability and aggregation of protein using ribonuclease, bovine serum albumin and lysozyme using temperature-regulated circular dichroism (CD) and differential scanning microcalorimetry (DSC). Ribonuclease showed no change in thermal stability and aggregation by the addition of allantoin. While allantoin showed no effects on the thermal stability of bovine serum albumin, it enhanced aggregation. Similarly, allantoin showed no stabilizing effects on lysozyme, but it strongly suppressed aggregation. Such suppressed aggregation resulted in reversibility of thermal unfolding of lysozyme. These effects of allantoin were then compared with those of NaCl and arginine hydrochloride. Arginine was similar to allantoin at low concentrations, where both solvent additives can be compared due to limited solubility of allantoin. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Allantoin, an inexpensive natural product, is a cell metabolite present in a variety of plants, including wheat germs, tobacco seeds and comfrey roots [1,2]. Allantoin is also found in animals and can be chemically synthesized from urea [3]. Allantoin has antiinflammatory, anti-ulcer and cell-growth promoting activities and is thus widely used in a variety of commercial non-pharmaceutical (e.g. cosmetic), and pharmaceutical (e.g. as a wound healing skin care agent) [4–9]. It is slightly soluble in aqueous solution and readily forms crystals, and is chemically unstable in alkaline solution. Recently, a novel application of crystalline allantoin has been reported by Gagnon et al. [10–15]. Allantoin crystals were found to bind endotoxins and high molecular weight aggregates, which contain variable amounts of chromatin, in cell culture harvests with high specificity and affinity. To our knowledge, no studies have been reported on the effects of soluble allantoin on protein solution. Allantoin is chiral as shown in Fig. 1 (circle) and contains amide groups and as such, its structure is similar to amine-containing low molecular weight compounds that have been shown to suppress protein aggregation during thermal melting [16–18]. Arginine has
∗ Corresponding author at: Alliance Protein Laboratories, 6042 Cornerstone Court West A, San Diego, CA, 92121, United States. E-mail address: [email protected] (T. Arakawa).
also been found to be a highly effective aggregation suppressor at high concentration. Compared with the synthetic amine compounds, arginine is a natural amino acid and thus safe to be used as an aggregation suppressing agent [19–23]. In this paper, we have investigated the effects of soluble allantoin on heat-induced structure changes and resultant aggregation of globular proteins and compared the results with the effects of NaCl and arginine.
2. Materials and methods Three commercial proteins, BSA (bovine serum albumin), Lyso (lysozyme) and RNase (ribonuclease), were used as received. These proteins were dissolved in phosphate-buffered saline (PBS, 10 mM phosphate, 0.15 M NaCl, pH 7.0) at 20 mg/ml and used as a stock solution. A stock solution of allantoin was prepared at 200 mM in PBS by heating at 95 ◦ C. Once dissolved, the stock solution was kept at 75 ◦ C without precipitation. The stock solution was prepared daily. Stock solution containing 1 M arginine or 2 M NaCl were also prepared in PBS. Circular dichroism (CD) analysis was carried out on a Jasco J-1500 spectropolarimeter equipped with a Peltier temperature controller. Since arginine and allantoin have strong absorbance in the far UV region, the near UV region was used for CD measurements. Near UV CD spectra and melting curves were determined using 1 cm path-length quartz cell with 1 cm width. A 2 mL aliquot
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2
66 65
NaCl
64 Fig. 1. Structure of allantoin.
Asymmetric carbon is circled.
63
Arginine
62 61 0
500
1000
1500
Fig. 3. Melting temperature of RNase as a function of allantoin (square), arginine (diamond) and NaCl (triangle) concentration. Melting of 4 mg/ml BSA was followed at 270 nm.
Fig. 2. Thermal melting of RNase in the absence (curve 1) and presence of 0.9 M arginine (curve 2) and 1.8 M NaCl (curve 3). Melting of 2 mg/ml RNase was followed at 276 nm.
of the samples was loaded in the cell. Melting was followed at the indicated wavelength with a temperature increment of 1 ◦ C/min. The sample and reference cells (made of tantalum) were loaded with ∼0.5 mL of degassed sample and buffer, respectively. A MicroCal VP-DSC was programmed to scan from 10 to 95 ◦ C at a rate of 60 ◦ C/hr with an 8 s filtering period and no feedback. Several buffer vs. buffer scans were recorded before running the sample vs. buffer experiment to bring the thermal environment of the instrument to a steady state and to obtain a pre-experiment buffer-buffer baseline. After each sample-buffer scan, the cell was cleaned in cycle with 10% Contrad, followed by an exhaustive water wash. The raw data were processed and analyzed using Origin 7.0. A buffer-buffer scan was subtracted from each sample-buffer scan, and the baseline was calculated and processed using the Origin software according to the manufacture’s instructions. The baseline-corrected Cp profiles were normalized to protein concentration (expressed as kcal/mol/◦ C). 3. Results and discussion 3.1. Ribonuclease The near UV CD spectra of 1.5 mg/ml RNase in PBS were compared at 20 and 90 ◦ C (data not shown), from which the optimal protein concentration and wavelength to monitor thermal melting were determined to be 2 mg/ml and 276 nm. Fig. 2 shows the change in CD intensity at 276 nm of 2 mg/ml RNase in PBS with increasing temperature from 40 to 80 ◦ C (curve 1). The negative CD signal at this wavelength started increasing at 57 ◦ C with a transition mid-temperature (Tm) of ∼64 ◦ C. This melting was accompanied by no increase in HT[V] signal at 276 nm (data not shown), which closely follows the absorbance property and hence the light scattering of the sample. It thus does not appear that ther-
mal unfolding of RNase accompanies aggregation. Fig. 2 also shows the melting curves of RNase in 0.9 M arginine (curve 2) and 1.8 NaCl (curve 3). The melting curve was shifted to a higher temperature in NaCl and to a lower temperature in arginine, indicating stabilization by NaCl and destabilization by arginine of RNase structure. Neither case involved increase in HT[V] intensity, indicating that neither NaCl nor arginine caused visible aggregation of RNase, as in the buffer solution. Fig. 3 plots the melting temperature of RNase as a function of additive concentration. It is evident that both arginine and NaCl had no effects on melting temperature at low concentrations and that at higher concentration NaCl increased the melting temperature while arginine decreased it. Allantion showed no effects on melting temperature and HT[V] signals of RNase, although higher concentration of allantoin could not be used due to its limited solubility. It may be summarized here that RNase does not appear to aggregate upon melting and that NaCl and arginine have an opposite effect on its thermal stability. Allantoin is inert to thermal stability and aggregation of RNase. 3.2. BSA Similarly, the near UV CD spectra of 2 mg/ml BSA in PBS were compared at 30 and 90 ◦ C (data not shown), from which the optimal wavelength to monitor BSA melting was determined to be 270 nm. Fig. 4A shows the melting of 4 mg/ml BSA in PBS (curve 1). The CD intensity at 270 nm started increasing at ∼55 ◦ C with a Tm of 65 ◦ C. In this case, the HT[V] intensity started increasing around 55 ◦ C as shown in Fig. 4B (curve 1), reflecting thermal unfolding of BSA. Fig. 4A also shows the melting curves of 4 mg/ml BSA in 0.8 M arginine (curve 2) and 0.8 M NaCl (curve 3). Both additives shifted the melting curves to higher temperature, stabilizing the BSA. Fig. 5 plots the Tm against arginine (diamonds) and NaCl (triangles) concentration. While NaCl linearly increased the Tm, arginine appeared to level-off at 500 mM. Fig. 5 also shows the effects of allantoin (squares), which resulted in no changes in Tm up to 100 mM. BSA at 4 mg/ml showed aggregation above 55 ◦ C as described above (Fig. 4B). Fig. 4B also shows the HT[V] signal at 270 nm as a function of temperature in 10 mM (curve 2) and 40 mM (curve 3) allantoin. It is evident that the rate of HT[V] increase was greater in the presence of allantoin, more so at 40 mM and thus that allantoin enhanced aggregation of BSA. The difference in HT[V] (HT[V]) between 40 and 80 ◦ C is plotted in Fig. 6 as a function of arginine (diamonds), NaCl (triangles) and allantoin (squares) concentration. Allantoin resulted in a steady increase in HT[V], indicating its enhancing effects on BSA aggregation. Arginine (diamond) initially followed the same trend, sharply increasing HT[V]. However, the
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Fig. 4. Thermal melting (A) and HT[V] of BSA (B). A: No additive (curve 1), 0.8 M arginine (curve 2), 0.8 M NaCl (curve 3). B: No additive (curve 1), 10 mM allantoin (curve 2), 40 mM allantoin (curve 3).
the three additives showed a different behavior on BSA. Allantoin showed no effects on melting temperature, but enhanced aggregation. Arginine increased the melting temperature slightly and enhanced aggregation at low concentration, but reduced aggregation at high concentration. NaCl showed weak enhancement of BSA aggregation, while significantly increasing the melting temperature.
69 68 67 66
3.3. Lysozyme
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Fig. 5. Melting temperature of BSA as a function of allantoin (square), arginine (diamond) and NaCl (triangle) concentration.
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Fig. 6. HT[V] of BSA as a function of allantoin (square), arginine (diamond) and NaCl (triangle) concentration.
HT[V] reached a plateau around 100 mM arginine, after which the HT[V] gradually decreased with increasing arginine concentration, eventually leading to a value smaller than in the absence of arginine. NaCl (triangles) resulted in a small increase in HT[V] both at low and high NaCl concentrations. An interesting observation is that the increase in HT[V] was smaller in NaCl than arginine at 100 mM, but greater in NaCl at 800 mM. It is thus concluded that
Comparison of the near UV CD spectra of 1 mg/ml Lyso in PBS at 25 and 90 ◦ C indicated the optimal wavelength to be 288 nm. Fig. 7A shows the melting curve of Lyso in PBS (curve 1), indicating a commencement of melting at 68 ◦ C with a Tm of 73–74 ◦ C. The melting temperatures were unchanged in the presence of allantoin, arginine and NaCl all up to 100 mM (data not shown). Namely, they do not affect the thermal stability of Lysozyme, perhaps due to its inherent high thermal stability. Fig. 7B shows the change in HT[V] at 288 nm with increasing temperature in the absence (curve 1) and presence of 5 mM (curve 2), 10 mM (curve 3) and 100 mM (curve 4) allantoin. The HT[V] signal at 288 nm started increasing around 75 ◦ C followed by a sharp increase, indicating extensive aggregation. The rate of HT[V] increase was slowed considerably with increasing allantoin concentration, as clearly seen in Fig. 8 (squares). At 100 mM, allantoin appeared to fully suppress Lyso aggregation, as HT[V] was close to zero. This was confirmed by demonstrating that Lyso unfolding under these conditions was fully reversible. Fig. 7A shows a second thermal scan (curve 2) after heating to 92 ◦ C in the first scan, indicating full reversibility by this technique. Fig. 8 also shows HT[V] in arginine (diamonds) and NaCl (triangles). There were no changes in NaCl, indicating that NaCl neither increased nor decreased thermal aggregation of Lysozyme. Arginine (diamonds) showed a similar trend to allantoin, suppressing thermal aggregation. It may be concluded that while allantoin, arginine and NaCl showed no effects on Lyso thermal stability, allantoin and arginine showed strong suppression on thermal aggregation of Lysozyme. The effect of allantoin on the thermal denaturation properties of Lyso was also studied using DSC, which can provide more detailed information on thermal unfolding. Fig. 9 shows the heat capacity profiles for the thermal denaturation of a 1 mg/ml Lysozyme solution in the absence and presence of 100 mM allantoin. Fig. 9 shows the raw heat capacity data (i.e. not baseline corrected), while
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Fig. 7. Thermal melting (A) and HT[V] (B) of Lyso. Melting of 1 mg/ml Lyso was followed at 288 nm. A: scan 1 (curve 1), scan 2 (curve 2). B: non additive (curve 1), 5 mM allantoin (curve 2), 10 mM allantoin (curve 3), 100 mM allantoin (curve 4).
lysozyme, scan 1 lysozyme + allantoin, scan 1 lysozyme + allantoin, scan 2
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Fig. 8. HT[V] of Lyso as a function of allantoin (square), arginine (diamond) and NaCl (triangle) concentration.
0 55
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Fig. 10. Baseline-corrected heat capacity profile. Curve 1: no additive. Curve 2: scan 1 with 100 mM allantoin. Curve 3: scan 3 with 100 mM allantoin.
Fig. 9. Heat capacity profile of Lyso. Curve B, buffer. Curve 1 (scan 1) and 2 (scan 2): no additive. Curve 3 (scan 1), 4 (scan 2), 5 (scan 3) and 6 (scan 4): 100 mM allantoin.
Fig. 10 shows baseline corrected data for selected scans. In Fig. 9, the red trace (curve 1) corresponds to the denaturation of Lyso (in the absence of allantoin), and gives a Tm of 72.84 ◦ C, which is consistent with the CD melting result. Note that the shift to lower heat capacity values from the buffer-buffer scan (curve B) to the lysozyme-buffer scan is due to the displacement of buffer by protein, since buffer (which is mostly water) has a higher heat capacity than protein. The blue trace (curve 2) corresponds to a repeat scan of the same material; after completion of the first denaturation scan, the instrument rapidly cooled the sample cell from the highest temperature of 95 ◦ C down to 10 ◦ C, held it at that temperature for 15 min, and then initiated the second thermal denaturation scan. Thus, this experiment investigated the extent to which Lyso would reversibly fold into the native structure after thermal denaturation. The blue trace (curve 2) shows that under these conditions and given these particular denaturation parameters, its thermal denaturation is completely irreversible, consistent with the observed extensive aggregation from the CD experiments (Fig. 7B, curve 1).
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When this experiment was carried out in the presence of 100 mM allantoin (the green trace, curve 3, representing the first scan), we can see that approximately 40% of the total protein was able to refold under these conditions (scan 2, curve 4, shown in magenta), and again for scan 3 (the cyan trace, curve 5) a small amount of heat was detected, indicating that a small amount of refolding occurred. The orange trace (curve 6) shows that no observable refolding was detected for scan 4. Thus, allantoin promotes the refolding of thermally denatured Lyso, consistent with the CD experiments. However, in contrast to the CD experiment, we did not observe 100% refolding, which may be due to the fact that the heating and cooling parameters between the two techniques were not identical and that thermally denatured Lyso were exposed to different cell surface (quartz cell for CD and tantalum cell for DSC). The Tm for unfolding of Lyso in the presence of 100 mM alantoin was 72.97 ◦ C and 72.61 ◦ C for scans 1 and 2, respectively (see Fig. 10), which are essentially indistinguishable from each other. However, the difference in the total unfolding enthalpy observed between the first thermal denaturation scans for Lyso in the absence and presence of allantoin is almost certainly outside the normal variability observed for this technique, and suggests that allantoin does affect the total heat flow observed during the melting process. The total unfolding enthalpies for the baseline corrected scans shown in Fig. 10 (the total area under the curve) are 90 kcal/mol, 73 kcal/mol and 27 kcal/mol for lysozyme alone, Lyso + allantoin (scan 1) and lysozyme + allantoin (scan 2), respectively. While no change in melting temperature by allantoin suggests its binding to both native and unfolded states of Lysozyme, it is not clear why the enthalpy of unfolding was lower in the presence of allantoin. 4. Conclusion Three proteins were used to examine the effects of allantoin, arginine and NaCl on thermal aggregation in PBS. First, RNase did not appear to aggregate upon melting in PBS. NaCl and arginine had opposite effects on its thermal stability. Allantoin was inert to thermal stability and aggregation of RNase. These 3 additives showed a different behavior on BSA. Allantoin showed no effects on melting temperature, but enhanced aggregation. Arginine increased the melting temperature slightly and enhanced aggregation at low concentration, but reduced aggregation at high concentration. NaCl showed weak enhancement of BSA aggregation, while significantly increasing the melting temperature. Against lysozyme, allantoin, arginine and NaCl showed no effects on thermal stability. On the contrary, allantoin and arginine showed strong suppression on thermal aggregation. Allantoin at 100 mM resulted in reversible thermal melting.
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