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Effects of the concentration and heating rate on the thermal denaturation and reversibility of granulocyte-colony stimulating factor studied by circular dichroism and infrared spectroscopy
Vibrational Spectroscopy 38 (2005) 33–38 www.elsevier.com/locate/vibspec
Effects of the concentration and heating rate on the thermal denaturation and reversibility of granulocyte-colony stimulating factor studied by circular dichroism and infrared spectroscopy Katsuyoshi Yamazaki a,c, Takafumi Iwura a, Koichi Murayama b, Rika Ishikawa a, Yukihiro Ozaki c,* a
Product Development Section, CMC R&D Laboratories, Pharmaceutical Division, Kirin Brewery Co., Ltd., Takasaki-shi, Gunma 370-0013, Japan b Department of Biochemistry and Biophysics, Gifu University School of Medicine, Gifu 500-8705, Japan c Department of Chemistry, School of Science and Technology, Kwansei-Gakuin University, Sanda 669-1337, Japan Available online 16 March 2005
Abstract Temperature-dependent (25–90 8C) circular dichroism (CD) spectra were measured for recombinant methionyl human granulocytecolony stimulating factor (rmethuG-CSF) in aqueous solutions over a pH range of 2.0–5.0 to investigate its thermal stability and reversibility. The effects of the protein concentration and heating rate on the thermal denaturation and reversibility were studied. We also compared the melting temperature (Tm) of rmethuG-CSF obtained from the CD spectra with that from the infrared (IR) spectra. The CD study of rmethuGCSF with the concentration of 0.5 mg/mL has demonstrated that rmethuG-CSF is the most stable at pH 2.8 in the pH range of 2.0–5.0 when the heating rate is 1 8C/min. This result does not always agree with the IR result, obtained using the concentration of 10 mg/mL, that the secondary structure of rmethuG-CSF is the most stable at pD 2.5 in the pD range of 2.5–5.5. However, when the CD measurements are curried out for the protein concentration of 5.0 mg/mL and the heating rate of 0.2 8C/min, which are similar measurement conditions to those for the IR study, Tm obtained from the CD data is almost identical with that from the IR data. The reversibility of rmethuG-CSF has been found to be the highest at pH 2.0 by the CD analysis. It seems that the Tm of rmethuG-CSF is easily affected by the measurement conditions such as protein concentration and heating rate as its reversibility is low. The CD study have also revealed that as pH is increased b-structure is increased after heating. # 2005 Elsevier B.V. All rights reserved. Keywords: Protein; Granulocyte-colony stimulating factor; CD; Thermal stability; Thermal denaturation
1. Introduction Granulocyte-colony stimulating factor (G-CSF) is a cytokine that plays a critical role in maintaining the population of neutrophilic granulocytes in peripheral blood and is also responsible for granulocytosis during inflammation [1,2]. Recombinant methionyl human G-CSF (rmethuG-CSF) overexpressed in Escherichia coli is composed of a polypeptide chain of 175 amino acids, and * Corresponding author. Tel.: +81 79 565 8349; fax: +81 79 565 9077. E-mail address: [email protected] (Y. Ozaki). 0924-2031/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2005.02.004
its molecular weight is approximately 19 kD. RmethuGCSF is a pharmaceutically relevant globular protein belonging to a group of growth factors that share the common four-helix bundle architecture [3]. Infrared (IR), circular dichroism (CD) and fluorescence studies of rmethuG-CSF have demonstrated that rmethuG-CSF undergoes structural changes induced by pH, heat and denaturant such as guanidine hydrochloride [4–6]. It has been shown by IR, CD and fluorescence studies that the secondary and tertiary structure of rmethuG-CSF is stable at low pH [6]. At pH 2.0, rmethuG-CSF exists in a compact state with a welldefined tertiary structure that unfolds in a cooperative
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K. Yamazaki et al. / Vibrational Spectroscopy 38 (2005) 33–38
fashion upon heating and is very stable against heat-induced unfolding [6]. The melting temperature (Tm) can be determined by various calorimetric and spectroscopic techniques. When the thermal stability of a protein is assessed by using Tm, one should take into account measurement conditions such as protein concentration and heating rate. Especially, when the unfolding reaction is at least partially irreversible, the differences in the measurement conditions influence on the evaluation of Tm. The purpose of the present study is to explore influences of the reversibility and measurement conditions of rmethuGCSF on the thermal stability evaluated by Tm. We measured far-ultraviolet (UV) CD spectra of rmethuG-CSF in aqueous solutions over a temperature range of 25–90 8C in the pH range of 2.0–5.0 and their IR spectra over a temperature range of 25–80 8C in the pD range of 2.5–5.5. The concentrations of rmethuG-CSF used for the CD and IR measurements were 0.5 and 10 mg/mL, respectively. By use of the obtained spectra the Tm and reversibility of rmethuGCSF were determined, and the fraction of each secondary structure element was estimated.
CD spectrum was measured. The intensity of a signal at 222 nm regained during the 25 8C incubation relative to that of the initial signal was measured, and then used to determine the percentage of the reversibility. 2.3. Infrared spectroscopy
Recombinant human G-CSF overexpressed in E. coli was produced and purified at KIRIN BREWERY Co., Ltd. (Gunma, Japan). The concentration of rmethuG-CSF was determined spectrophotometrically using the extinction coefficient of 0.86 at 280 nm for a 0.1% (w/v) solution of the protein.
A rmethuG-CSF solution with the concentration of 10 mg/mL was exchanged from a HCl solution (pH 4.0) into a 20 mmol/L Na phosphate buffer at pD 2.5, 3.0, 3.5, 4.0, 5.0 and 5.5. IR spectra of the rmethuG-CSF solutions and buffers at the above pH were measured by using a Nicolet NEXUS 670 FT-IR spectrophotometer equipped with a liquid nitrogen cooled mercury cadmium telluride (MCT) detector and a temperature controller having a water bus (Thermo Haake DC30). A sample solution was placed between CaF2 windows with a 50 mm Teflon spacer. For each spectrum, 256 scans were coadded with a spectral resolution of 2 cm 1. The sample chamber of the spectrometer was continuously purged with N2 gas to prevent atmospheric water vapor from obscuring amide I and II regions. The spectra were measured over a temperature range of 25–80 8C with an increment of approximately 0.3 8C/min. Spectral subtraction was performed using OMNIC program (Thermo Nicolet). The IR spectrum of atmospheric water vapor was subtracted from each spectrum, and then, the spectrum of a buffer solution of particular pH was subtracted from the spectrum of the corresponding protein solution obtained under the same conditions. The spectra thus obtained were subjected to smoothing. The smoothing and the calculation of the second-derivative spectra and curve fitting spectra were made by using homemade software named SPINA 3.0 (Dr. Y. Katsumoto, Kwansei-Gakuin University).
2.2. Circular dichroism spectroscopy, unfolding studies and reversibility
3. Results
2. Materials and methods 2.1. Materials
Far-UV CD spectra in the 250–200 nm region were measured for rmethuG-CSF in a 20 mmol/L Na phosphate buffer with the concentration of 0.5 and 5 mg/mL. A Jasco J-820 spectropolarimeter and thermal control (Jasco CDF426L) were used for the CD measurements, and a cell of 1 or 0.1 mm path-length was employed. At each pH the buffer spectrum was subtracted from the corresponding protein spectrum. For the study of thermal unfolding of rmethuG-CSF, the sample temperature was increased at 0.2 and 1 8C/min and the CD signal at 222 nm was monitored. The fraction of the folded protein was calculated by the method previously reported [7,8], and plotted against temperature to give unfolding curves. To determine the degree of reversibility a rmethuG-CSF buffer solution was cooled down to 25 8C immediately after the temperature of the solution reached at 90 8C, and the protein solution was incubated at 25 8C for 15 min. Then, its
3.1. pH dependence of thermal denaturation of rmethuG-CSF The thermal stability of secondary structure of rmethuGCSF was investigated in the pH range from 2.0 to 5.0 by means of far-UV CD. The study was performed using the protein concentration of 0.5 mg/mL and the heating rate of 1 8C/min. The thermal stability was assessed by the midpoint of the thermal transition, Tm. Fig. 1a shows the thermally induced denaturation curves of rmethuG-CSF for the pH range from 2.0 to 5.0. The Tm obtained for the buffer solutions in the pH range of 2.0–5.0 is summarized in Table 1. Fig. 1b plots the Tm as a function of pH. Note that the plots show a peak maximum at pH 2.8 (Tm = 68.3 8C). Therefore, it seems that rmethuG-CSF (0.5 mg/mL) is the most stable at pH 2.8. The thermal denaturation at pH 5.0 was accompanied by the precipitation of protein. At other pH, no precipitation was observed.
K. Yamazaki et al. / Vibrational Spectroscopy 38 (2005) 33–38
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Fig. 2. Plots of the reversibility of rmethuG-CSF at pH 2.0, 2.5, 2.6, 2.8, 3.0, 3.2, 4.0 and 5.0.
of rmethuG-CSF is the highest at pH 2.8 while its reversibility is the highest at pH 2.0 in the pH range of 2.0–5.0. Thus, the condition where Tm is the highest is different from that where the reversibility is the highest. 3.3. Effects of the protein concentration on Tm and reversibility
Fig. 1. (a) Thermal unfolding curves for rmethuG-CSF at pH 2.0, 2.5, 3.0, 4.0 and 5.0. (b) Plots of the melting temperature (Tm) of rmethuG-CSF at pH 2.0, 2.5, 2.6, 2.8, 3.0, 3.2, 4.0 and 5.0.
3.2. Reversibility The reversibility of secondary structure of rmethuG-CSF was measured by using far-UV CD in the pH range from 2.0 to 5.0; the results are summarized in Table 1. Fig. 2 plots the reversibility as a function of pH. As pH is decreased, the degree of reversibility becomes higher. Approximately, 90% of the molecular ellipticity at 222 nm recovered after cooling the protein solution from 90 to 25 8C at pH 2.0. At pH 5.0, the reversibility was only 10%, yielding the precipitation, which means that rmethuG-CSF has little native secondary structure. Under other pH conditions, the protein remains soluble. Interestingly, the thermal stability Table 1 pH dependence of the Tm and reversibility of thermal denaturation of rmethuG-CSF pH
Tm (8C)
Reversibility (%)
Precipitation observed
2.0 2.5 2.6 2.8 3.0 3.2 4.0 5.0
62.3 65.8 66.6 68.3 67.8 66.7 63.9 58.8
90.9 84.9 86.2 78.0 63.5 60.5 58.1 11.8
No No No No No No No Yes
We studied the effects of the concentration of rmethuGCSF on the Tm and the degree of reversibility of the thermal denaturation. The protein solutions with the concentration of 0.5 and 5.0 mg/mL were heated at 1 8C/min at pH 2.5 and 4.0. The results are shown in Table 2. The increase in the protein concentration from 0.5 to 5 mg/mL at pH 2.5 affects little the Tm, but does affect the reversibility. The increase in the protein concentration at pH 4.0 affects little the reversibility, but does affect the Tm, decreasing it by approximately 4 8C. The change in the protein concentration at pH 4.0 has the clearer influence in the Tm than that at pH 2.5. 3.4. Effect of heating rate on the thermal denaturation To explore the effects of heating rate on the thermal denaturation of rmethuG-CSF the protein aqueous solutions with the concentration of 0.5 mg/mL at pH 2.5 and 4.0 were heated with the heating rates of 1 and 0.2 8C/min. The results are shown in Table 2. Of note is that the decrease in the heating rate at pH 4.0 lowers the Tm, but at pH 2.5 it affects the Tm a little and that the decrease in the heating rate reduces the reversibility at both pH. A slower heating rate leads to the protein aggregation. After heating the protein solution of 0.5 mg/mL at 1 8C/min at pH 2.5, 85% of rmethuG-CSF recovered while after heating at 0.2 8C/min, 72% recovered. After heating the solution of 0.5 mg/mL at 1 and 0.2 8C/min at pH 4.0, the reversibility was 58 and 34%, respectively. It seems that the degree of the decrease in the reversibility at pH 4.0 is higher than that at pH 2.5, when the heating rate is changed from 1 to 0.2 8C/min. Therefore, the
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Table 2 Effects of the concentration and heating rate of rmethuG-CSF on the thermal denaturation and the comparison of results obtained by CD and IR pH
Protein concentration (mg/mL)
Heating rate (8C/min)
Method
Tm (8C)
Reversibility (%)
2.5 2.5 2.5 2.5 2.5 4.0 4.0 4.0 4.0 4.0
0.5 0.5 5.0 5.0 10 0.5 0.5 5.0 5.0 10
1 0.2 1 0.2 0.3 1 0.2 1 0.2 0.3
CD CD CD CD IR CD CD CD CD IR
65.8 65.7 66.7 63.9 62 63.9 61.1 60.1 55.2 54
84.9 71.7 62.1 57.3 – 58.1 34.0 57.3 22.1 –
slower heating rate tends to affect the reversibility more clearly at pH 4.0 than at pH 2.5. The change of heating rate at pH 4.0 has strong influences on the Tm and reversibility than that at pH 2.5. 3.5. Comparison of the CD data with the IR data We studied the thermal stability of rmethuG-CSF over a pD range of 2.5–5.5 also using IR spectroscopy. It was found that the thermal stability of secondary structure increases and the protein aggregation does not tend to form as pH is decreased. The secondary structure of rmethuG-CSF seems to be the most stable at pD 2.5 over a pD range of 5.5–2.5. Note that the result of thermal stability obtained by IR spectroscopy is not always consistent with that of the CD spectra. The CD data show that the protein is the most stable at pH 2.8 as mentioned above. The reason for this discrepancy is caused by the differences in the protein concentration and the heating rate between the IR and CD measurements of rmethuG-CSF. The protein concentrations used for the IR and CD analyses are 10 and 0.5 mg/mL, respectively. The heating rates used for the IR and CD measurements are approximately 0.3 and 1 8C/min, respectively. When the CD spectra were measured at pH 2.5 and 4.0 under the conditions that the protein concentration was 5 mg/mL and the heating rate was 0.2 8C/min, the Tm obtained from the CD data is almost the same as that from the IR data (Table 2). The results indicate that rmethuG-CSF at pH 4.0 is more influenced by the changes in the conditions of measurements than that at pH 2.5.
summarizes the percentages of secondary structure elements of rmethuG-CSF before and after heating in the pH range of 2.0–5.0. The far-UV CD spectra of rmethuG-CSF before heating demonstrate that the amount of a-helix is approximately 70% through the pH range of 2.0–5.0 and that b-structure is not included. Interestingly, the far-UV CD spectra after heating show that the a-helix decreases and the b-structure increases as pH is increased to 4.0. After heating the a-helix decreases from 70 to 35% at pH 4.0, while the bstructure and random coil increase. Especially, the bstructure content reaches 30%. The b-structure seems to be intermolecular b-structure derived from the aggregation. The profile of the change of secondary structure composition before and after heating is very similar to the result of reversibility as mentioned above. Compared with those at pH 4.0, both a-helix and b-structure contents apparently decrease at pH 5.0. As mentioned above, the precipitation occurred at pH 5.0 after heating. The soluble proteins remained little in the solution by the formation of precipitates, and hence the signal of CD spectrum recovered little. Therefore, one cannot estimate accurately the amount of each secondary structure in the protein solution after heating at pH 5.0. However, it may be concluded from the formation of precipitates that rmethuG-CSF at pH 5.0 after heating has little native secondary structure.
3.6. Estimation of secondary structure elements of rmethuG-CSF before and after heating We estimated the percentages of secondary structure elements of rmethuG-CSF before and after heating by using the far-UV CD data. The relative amounts of secondary structure elements were calculated by the method of Yang et al. [9]. In this method, the fractions of the a-helix, b-form, b-turn and unordered form can be estimated by a leastsquares method using reference CD spectra based on fifteen proteins [9]. Fig. 3 plots the percentages of a-helix and b-structure versus pH before and after heating. Table 3
Fig. 3. Plots of the percentages of a-helix and b-structure before and after heating as a function of pH: (*) a-helix before heating; (*) a-helix after heating; (&) b-structure before heating; (&) b-structure after heating.
K. Yamazaki et al. / Vibrational Spectroscopy 38 (2005) 33–38
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Table 3 Fractions of secondary structure elements of rmethuG-CSF before and after heating (%) pH
Before
After
a-Helix
b-Structure
Turn
Random
a-Helix
b-Structure
Turn
Random
2.0 2.5 2.6 2.8 3.0 3.2 4.0 5.0
68.8 68.1 67.4 67.5 70.8 71.4 68.5 67.6
0 0 0 0 0 0 0 0
5.6 5.8 6.6 6.4 4.1 3.5 5.7 6.4
25.6 26.1 25.9 26.1 25.1 25.1 25.8 26.0
60.6 55.2 54.9 46.6 38.5 35.9 35.7 17.1
3.8 8.3 8.6 21.9 27.3 28.2 31.8 9.0
6.0 5.5 5.3 0.0 0.0 0.0 4.0 43.4
29.6 30.9 31.2 31.5 34.2 35.9 28.4 30.4
4. Discussion In the present study, we have investigated the Tm and reversibility of rmethuG-CSF by means of far-UV CD and compared the Tm obtained from the CD measurements with that from the IR study. According to the CD studies, the secondary structure of rmethuG-CSF is the most stable at pH 2.8, but its reversibility is the highest not at this pH but at pH 2.0. The result indicates that the net charge of rmethuG-CSF at pH 2.8 makes the conformation stable. RmethuG-CSF at pH 2.0 is more positively charged, resulting in the stronger protein–protein electrostatic repulsion. Once the conformation of rmethuG-CSF partially unfolds by heating, the electrostatic repulsion suppresses to form the aggregates. Balance between the effect of conformation stability and that of electrostatic repulsion determines the thermal stability of rmethuG-CSF. Border pH for this balance seems to be pH 2.8 under the conditions of our CD study. Recent studies of Chi et al. suggested that the aggregation of rmethuG-CSF is controlled by both conformation stability and colloidal stability and depends on the solution conditions [11]. Our conclusion is supported by the results of Chi et al. Generally, the protein concentration of more than 10 mg/ mL is required to obtain IR spectra. For far-UV CD analysis, the protein concentration of 0.1–1 mg/mL is usually required. Therefore, the region of protein concentration required for the measurements is different between IR and CD. In the thermal stability study of rmethuG-CSF, rmethuG-CSF is more easily influenced at pH 4.0 by the measurement conditions than at pH 2.5. The Tm of rmethuGCSF is easily affected by the measurement conditions such as protein concentration and heating rate as the reversibility is low. We cannot measure a CD spectrum of a protein with the concentration of 10 mg/mL, which is the suitable concentration for the IR measurement, because of high CD absorbance of the protein. Accordingly, we measured the CD spectra of rmethuG-CSF with the concentration of 5 mg/mL. When the CD spectra of rmethuG-CSF are measured for the protein concentration of 5 mg/mL and the heating rate of 0.2 8C/min, the Tm obtained from CD is almost identical with that from IR. The thermal denaturation of rmethuG-CSF is not fully reversible. Its reversibility is approximately 60% at pH 4.0.
While the amount of a-helix decreases from 70 to 35% at pH 4.0, those of b-structure and random coil increase, especially b-structure content becomes 30%. The b-structure appears to be intermolecular antiparallel b-structure, leading to the aggregation. On the other hand, the IR spectrum of rmethuG-CSF at 25 8C shows a dominant band at 1654 cm 1 in the amide I region, indicative of a-helix. As temperature increases, the intensity of the band at 1654 cm 1 decreases, and instead new bands appear at 1685 and 1618 cm 1 (Fig. 4). The bands indicate that intermolecular antiparallel b-structure is formed by aggregation [10]. The aggregation is produced as the consequence of the thermal denaturation of rmethuG-CSF. The b-structure observed by CD after heating seems to be the same as the intermolecular antiparallel b-structure detected by the IR spectra. This study demonstrates that the amount of bstructure increases as pH increases over a pH range of 2.0– 4.0 and at pH 5.0 the precipitates are formed. This means that b-structure is soluble in the pH range of 2.0–4.0, but at pH 5.0 b-structure is beyond solubility limit and the precipitation is formed. Recently, Chi et al. demonstrated that the increase in the repulsive interactions between protein molecules is effective in reducing the aggregation in a rmethuG-CSF solution at low pH [11]. Therefore, the decrease in the repulsive interaction between denaturated proteins seems to cause the precipitation at pH 5.0.
Fig. 4. IR spectra in the amide I region of rmethuG-CSF in a 20 mmol/L Na phosphate buffer at pD 4.0 at 25 and 80 8C. Solid line represents the IR spectrum at 25 8C, while dashed line represents the IR spectrum at 80 8C.
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When the thermal stability of a protein is assessed using Tm, it is important to take into account measurement conditions such as protein concentration and heating rate. Especially, when the unfolding reaction is at least partially irreversible, the difference in the measurement conditions has more influence on the evaluation of Tm.
5. Conclusion The thermal stability of rmethuG-CSF is highest at pH 2.8 while its reversibility is the highest at pH 2.0 in the pH range of 2.0–5.0 under the measurement conditions of CD. The conditions where the Tm is the highest are different from those where the reversibility is the highest. RmethuG-CSF at pH 4.0 is more influenced by the changes in the measurement conditions such as the protein concentration and heating rate, than that at pH 2.5 because the reversibility of rmethuG-CSF at pH 2.5 is higher than that at pH 4.0. When the thermal stability of protein is assessed by using Tm, measurement conditions are very important. One of the purposes of the present study is to compare the results for the Tm obtained by CD and IR spectra. It was found that when the CD spectra of rmethuG-CSF were measured for the heating rate of 0.2 8C/min and the protein concentration of 5 mg/mL, which is pretty high for usual CD measurements for protein solution while low for usual IR measurements,
the Tm obtained from CD is almost identical with that from IR.
Acknowledgement We thank Associate Professor F. Arisaka (Tokyo Institute of Technology) for valuable advice on the analysis of the CD data.
References [1] N.A. Nicola, Annu. Rev. Biochem. 58 (1989) 45. [2] G.D. Demetri, J.D. Griffin, Blood 78 (1991) 2791. [3] C.P. Hill, T.D. Osslund, D. Eisenberg, Proc. Natl. Acad. Sci. 90 (1993) 5167. [4] L.O. Narhi, W.C. Kenney, T. Arakawa, J. Protein Chem. 10 (1991) 359. [5] C.G. Kolvenbach, S. Elliott, R. Sachdev, T. Arakawa, L.O. Narhi, J. Protein Chem. 12 (1993) 229. [6] C.G. Kolvenbach, L.O. Narhi, J.S. Philo, T.S. Li, M. Zhang, T. Arakawa, J. Pept. Res. 50 (1997) 310. [7] C.N. Pace, K.L. Shaw, Proteins Struct. Funct. Genet. Suppl. 4 (2000) 1. [8] C.N. Pace, Trends Biotechnol. 8 (1990) 93. [9] J.T. Yang, C.S. Wu, H.M. Martinez, Methods Enzymol. 130 (1986) 208. [10] A.L. Fink, Folding Des. 3 (1998) R9. [11] E.Y. Chi, S. Krishnan, B.S. Kendrick, B.S. Chang, J.F. Carpenter, T.W. Randolph, Protein Sci. 12 (2003) 903.
Effects of the concentration and heating rate on the thermal denaturation and reversibility of granulocyte-colony stimulating factor studied by circular dichroism and infrared spectroscopy Katsuyoshi Yamazaki a,c, Takafumi Iwura a, Koichi Murayama b, Rika Ishikawa a, Yukihiro Ozaki c,* a
Product Development Section, CMC R&D Laboratories, Pharmaceutical Division, Kirin Brewery Co., Ltd., Takasaki-shi, Gunma 370-0013, Japan b Department of Biochemistry and Biophysics, Gifu University School of Medicine, Gifu 500-8705, Japan c Department of Chemistry, School of Science and Technology, Kwansei-Gakuin University, Sanda 669-1337, Japan Available online 16 March 2005
Abstract Temperature-dependent (25–90 8C) circular dichroism (CD) spectra were measured for recombinant methionyl human granulocytecolony stimulating factor (rmethuG-CSF) in aqueous solutions over a pH range of 2.0–5.0 to investigate its thermal stability and reversibility. The effects of the protein concentration and heating rate on the thermal denaturation and reversibility were studied. We also compared the melting temperature (Tm) of rmethuG-CSF obtained from the CD spectra with that from the infrared (IR) spectra. The CD study of rmethuGCSF with the concentration of 0.5 mg/mL has demonstrated that rmethuG-CSF is the most stable at pH 2.8 in the pH range of 2.0–5.0 when the heating rate is 1 8C/min. This result does not always agree with the IR result, obtained using the concentration of 10 mg/mL, that the secondary structure of rmethuG-CSF is the most stable at pD 2.5 in the pD range of 2.5–5.5. However, when the CD measurements are curried out for the protein concentration of 5.0 mg/mL and the heating rate of 0.2 8C/min, which are similar measurement conditions to those for the IR study, Tm obtained from the CD data is almost identical with that from the IR data. The reversibility of rmethuG-CSF has been found to be the highest at pH 2.0 by the CD analysis. It seems that the Tm of rmethuG-CSF is easily affected by the measurement conditions such as protein concentration and heating rate as its reversibility is low. The CD study have also revealed that as pH is increased b-structure is increased after heating. # 2005 Elsevier B.V. All rights reserved. Keywords: Protein; Granulocyte-colony stimulating factor; CD; Thermal stability; Thermal denaturation
1. Introduction Granulocyte-colony stimulating factor (G-CSF) is a cytokine that plays a critical role in maintaining the population of neutrophilic granulocytes in peripheral blood and is also responsible for granulocytosis during inflammation [1,2]. Recombinant methionyl human G-CSF (rmethuG-CSF) overexpressed in Escherichia coli is composed of a polypeptide chain of 175 amino acids, and * Corresponding author. Tel.: +81 79 565 8349; fax: +81 79 565 9077. E-mail address: [email protected] (Y. Ozaki). 0924-2031/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2005.02.004
its molecular weight is approximately 19 kD. RmethuGCSF is a pharmaceutically relevant globular protein belonging to a group of growth factors that share the common four-helix bundle architecture [3]. Infrared (IR), circular dichroism (CD) and fluorescence studies of rmethuG-CSF have demonstrated that rmethuG-CSF undergoes structural changes induced by pH, heat and denaturant such as guanidine hydrochloride [4–6]. It has been shown by IR, CD and fluorescence studies that the secondary and tertiary structure of rmethuG-CSF is stable at low pH [6]. At pH 2.0, rmethuG-CSF exists in a compact state with a welldefined tertiary structure that unfolds in a cooperative
34
K. Yamazaki et al. / Vibrational Spectroscopy 38 (2005) 33–38
fashion upon heating and is very stable against heat-induced unfolding [6]. The melting temperature (Tm) can be determined by various calorimetric and spectroscopic techniques. When the thermal stability of a protein is assessed by using Tm, one should take into account measurement conditions such as protein concentration and heating rate. Especially, when the unfolding reaction is at least partially irreversible, the differences in the measurement conditions influence on the evaluation of Tm. The purpose of the present study is to explore influences of the reversibility and measurement conditions of rmethuGCSF on the thermal stability evaluated by Tm. We measured far-ultraviolet (UV) CD spectra of rmethuG-CSF in aqueous solutions over a temperature range of 25–90 8C in the pH range of 2.0–5.0 and their IR spectra over a temperature range of 25–80 8C in the pD range of 2.5–5.5. The concentrations of rmethuG-CSF used for the CD and IR measurements were 0.5 and 10 mg/mL, respectively. By use of the obtained spectra the Tm and reversibility of rmethuGCSF were determined, and the fraction of each secondary structure element was estimated.
CD spectrum was measured. The intensity of a signal at 222 nm regained during the 25 8C incubation relative to that of the initial signal was measured, and then used to determine the percentage of the reversibility. 2.3. Infrared spectroscopy
Recombinant human G-CSF overexpressed in E. coli was produced and purified at KIRIN BREWERY Co., Ltd. (Gunma, Japan). The concentration of rmethuG-CSF was determined spectrophotometrically using the extinction coefficient of 0.86 at 280 nm for a 0.1% (w/v) solution of the protein.
A rmethuG-CSF solution with the concentration of 10 mg/mL was exchanged from a HCl solution (pH 4.0) into a 20 mmol/L Na phosphate buffer at pD 2.5, 3.0, 3.5, 4.0, 5.0 and 5.5. IR spectra of the rmethuG-CSF solutions and buffers at the above pH were measured by using a Nicolet NEXUS 670 FT-IR spectrophotometer equipped with a liquid nitrogen cooled mercury cadmium telluride (MCT) detector and a temperature controller having a water bus (Thermo Haake DC30). A sample solution was placed between CaF2 windows with a 50 mm Teflon spacer. For each spectrum, 256 scans were coadded with a spectral resolution of 2 cm 1. The sample chamber of the spectrometer was continuously purged with N2 gas to prevent atmospheric water vapor from obscuring amide I and II regions. The spectra were measured over a temperature range of 25–80 8C with an increment of approximately 0.3 8C/min. Spectral subtraction was performed using OMNIC program (Thermo Nicolet). The IR spectrum of atmospheric water vapor was subtracted from each spectrum, and then, the spectrum of a buffer solution of particular pH was subtracted from the spectrum of the corresponding protein solution obtained under the same conditions. The spectra thus obtained were subjected to smoothing. The smoothing and the calculation of the second-derivative spectra and curve fitting spectra were made by using homemade software named SPINA 3.0 (Dr. Y. Katsumoto, Kwansei-Gakuin University).
2.2. Circular dichroism spectroscopy, unfolding studies and reversibility
3. Results
2. Materials and methods 2.1. Materials
Far-UV CD spectra in the 250–200 nm region were measured for rmethuG-CSF in a 20 mmol/L Na phosphate buffer with the concentration of 0.5 and 5 mg/mL. A Jasco J-820 spectropolarimeter and thermal control (Jasco CDF426L) were used for the CD measurements, and a cell of 1 or 0.1 mm path-length was employed. At each pH the buffer spectrum was subtracted from the corresponding protein spectrum. For the study of thermal unfolding of rmethuG-CSF, the sample temperature was increased at 0.2 and 1 8C/min and the CD signal at 222 nm was monitored. The fraction of the folded protein was calculated by the method previously reported [7,8], and plotted against temperature to give unfolding curves. To determine the degree of reversibility a rmethuG-CSF buffer solution was cooled down to 25 8C immediately after the temperature of the solution reached at 90 8C, and the protein solution was incubated at 25 8C for 15 min. Then, its
3.1. pH dependence of thermal denaturation of rmethuG-CSF The thermal stability of secondary structure of rmethuGCSF was investigated in the pH range from 2.0 to 5.0 by means of far-UV CD. The study was performed using the protein concentration of 0.5 mg/mL and the heating rate of 1 8C/min. The thermal stability was assessed by the midpoint of the thermal transition, Tm. Fig. 1a shows the thermally induced denaturation curves of rmethuG-CSF for the pH range from 2.0 to 5.0. The Tm obtained for the buffer solutions in the pH range of 2.0–5.0 is summarized in Table 1. Fig. 1b plots the Tm as a function of pH. Note that the plots show a peak maximum at pH 2.8 (Tm = 68.3 8C). Therefore, it seems that rmethuG-CSF (0.5 mg/mL) is the most stable at pH 2.8. The thermal denaturation at pH 5.0 was accompanied by the precipitation of protein. At other pH, no precipitation was observed.
K. Yamazaki et al. / Vibrational Spectroscopy 38 (2005) 33–38
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Fig. 2. Plots of the reversibility of rmethuG-CSF at pH 2.0, 2.5, 2.6, 2.8, 3.0, 3.2, 4.0 and 5.0.
of rmethuG-CSF is the highest at pH 2.8 while its reversibility is the highest at pH 2.0 in the pH range of 2.0–5.0. Thus, the condition where Tm is the highest is different from that where the reversibility is the highest. 3.3. Effects of the protein concentration on Tm and reversibility
Fig. 1. (a) Thermal unfolding curves for rmethuG-CSF at pH 2.0, 2.5, 3.0, 4.0 and 5.0. (b) Plots of the melting temperature (Tm) of rmethuG-CSF at pH 2.0, 2.5, 2.6, 2.8, 3.0, 3.2, 4.0 and 5.0.
3.2. Reversibility The reversibility of secondary structure of rmethuG-CSF was measured by using far-UV CD in the pH range from 2.0 to 5.0; the results are summarized in Table 1. Fig. 2 plots the reversibility as a function of pH. As pH is decreased, the degree of reversibility becomes higher. Approximately, 90% of the molecular ellipticity at 222 nm recovered after cooling the protein solution from 90 to 25 8C at pH 2.0. At pH 5.0, the reversibility was only 10%, yielding the precipitation, which means that rmethuG-CSF has little native secondary structure. Under other pH conditions, the protein remains soluble. Interestingly, the thermal stability Table 1 pH dependence of the Tm and reversibility of thermal denaturation of rmethuG-CSF pH
Tm (8C)
Reversibility (%)
Precipitation observed
2.0 2.5 2.6 2.8 3.0 3.2 4.0 5.0
62.3 65.8 66.6 68.3 67.8 66.7 63.9 58.8
90.9 84.9 86.2 78.0 63.5 60.5 58.1 11.8
No No No No No No No Yes
We studied the effects of the concentration of rmethuGCSF on the Tm and the degree of reversibility of the thermal denaturation. The protein solutions with the concentration of 0.5 and 5.0 mg/mL were heated at 1 8C/min at pH 2.5 and 4.0. The results are shown in Table 2. The increase in the protein concentration from 0.5 to 5 mg/mL at pH 2.5 affects little the Tm, but does affect the reversibility. The increase in the protein concentration at pH 4.0 affects little the reversibility, but does affect the Tm, decreasing it by approximately 4 8C. The change in the protein concentration at pH 4.0 has the clearer influence in the Tm than that at pH 2.5. 3.4. Effect of heating rate on the thermal denaturation To explore the effects of heating rate on the thermal denaturation of rmethuG-CSF the protein aqueous solutions with the concentration of 0.5 mg/mL at pH 2.5 and 4.0 were heated with the heating rates of 1 and 0.2 8C/min. The results are shown in Table 2. Of note is that the decrease in the heating rate at pH 4.0 lowers the Tm, but at pH 2.5 it affects the Tm a little and that the decrease in the heating rate reduces the reversibility at both pH. A slower heating rate leads to the protein aggregation. After heating the protein solution of 0.5 mg/mL at 1 8C/min at pH 2.5, 85% of rmethuG-CSF recovered while after heating at 0.2 8C/min, 72% recovered. After heating the solution of 0.5 mg/mL at 1 and 0.2 8C/min at pH 4.0, the reversibility was 58 and 34%, respectively. It seems that the degree of the decrease in the reversibility at pH 4.0 is higher than that at pH 2.5, when the heating rate is changed from 1 to 0.2 8C/min. Therefore, the
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Table 2 Effects of the concentration and heating rate of rmethuG-CSF on the thermal denaturation and the comparison of results obtained by CD and IR pH
Protein concentration (mg/mL)
Heating rate (8C/min)
Method
Tm (8C)
Reversibility (%)
2.5 2.5 2.5 2.5 2.5 4.0 4.0 4.0 4.0 4.0
0.5 0.5 5.0 5.0 10 0.5 0.5 5.0 5.0 10
1 0.2 1 0.2 0.3 1 0.2 1 0.2 0.3
CD CD CD CD IR CD CD CD CD IR
65.8 65.7 66.7 63.9 62 63.9 61.1 60.1 55.2 54
84.9 71.7 62.1 57.3 – 58.1 34.0 57.3 22.1 –
slower heating rate tends to affect the reversibility more clearly at pH 4.0 than at pH 2.5. The change of heating rate at pH 4.0 has strong influences on the Tm and reversibility than that at pH 2.5. 3.5. Comparison of the CD data with the IR data We studied the thermal stability of rmethuG-CSF over a pD range of 2.5–5.5 also using IR spectroscopy. It was found that the thermal stability of secondary structure increases and the protein aggregation does not tend to form as pH is decreased. The secondary structure of rmethuG-CSF seems to be the most stable at pD 2.5 over a pD range of 5.5–2.5. Note that the result of thermal stability obtained by IR spectroscopy is not always consistent with that of the CD spectra. The CD data show that the protein is the most stable at pH 2.8 as mentioned above. The reason for this discrepancy is caused by the differences in the protein concentration and the heating rate between the IR and CD measurements of rmethuG-CSF. The protein concentrations used for the IR and CD analyses are 10 and 0.5 mg/mL, respectively. The heating rates used for the IR and CD measurements are approximately 0.3 and 1 8C/min, respectively. When the CD spectra were measured at pH 2.5 and 4.0 under the conditions that the protein concentration was 5 mg/mL and the heating rate was 0.2 8C/min, the Tm obtained from the CD data is almost the same as that from the IR data (Table 2). The results indicate that rmethuG-CSF at pH 4.0 is more influenced by the changes in the conditions of measurements than that at pH 2.5.
summarizes the percentages of secondary structure elements of rmethuG-CSF before and after heating in the pH range of 2.0–5.0. The far-UV CD spectra of rmethuG-CSF before heating demonstrate that the amount of a-helix is approximately 70% through the pH range of 2.0–5.0 and that b-structure is not included. Interestingly, the far-UV CD spectra after heating show that the a-helix decreases and the b-structure increases as pH is increased to 4.0. After heating the a-helix decreases from 70 to 35% at pH 4.0, while the bstructure and random coil increase. Especially, the bstructure content reaches 30%. The b-structure seems to be intermolecular b-structure derived from the aggregation. The profile of the change of secondary structure composition before and after heating is very similar to the result of reversibility as mentioned above. Compared with those at pH 4.0, both a-helix and b-structure contents apparently decrease at pH 5.0. As mentioned above, the precipitation occurred at pH 5.0 after heating. The soluble proteins remained little in the solution by the formation of precipitates, and hence the signal of CD spectrum recovered little. Therefore, one cannot estimate accurately the amount of each secondary structure in the protein solution after heating at pH 5.0. However, it may be concluded from the formation of precipitates that rmethuG-CSF at pH 5.0 after heating has little native secondary structure.
3.6. Estimation of secondary structure elements of rmethuG-CSF before and after heating We estimated the percentages of secondary structure elements of rmethuG-CSF before and after heating by using the far-UV CD data. The relative amounts of secondary structure elements were calculated by the method of Yang et al. [9]. In this method, the fractions of the a-helix, b-form, b-turn and unordered form can be estimated by a leastsquares method using reference CD spectra based on fifteen proteins [9]. Fig. 3 plots the percentages of a-helix and b-structure versus pH before and after heating. Table 3
Fig. 3. Plots of the percentages of a-helix and b-structure before and after heating as a function of pH: (*) a-helix before heating; (*) a-helix after heating; (&) b-structure before heating; (&) b-structure after heating.
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Table 3 Fractions of secondary structure elements of rmethuG-CSF before and after heating (%) pH
Before
After
a-Helix
b-Structure
Turn
Random
a-Helix
b-Structure
Turn
Random
2.0 2.5 2.6 2.8 3.0 3.2 4.0 5.0
68.8 68.1 67.4 67.5 70.8 71.4 68.5 67.6
0 0 0 0 0 0 0 0
5.6 5.8 6.6 6.4 4.1 3.5 5.7 6.4
25.6 26.1 25.9 26.1 25.1 25.1 25.8 26.0
60.6 55.2 54.9 46.6 38.5 35.9 35.7 17.1
3.8 8.3 8.6 21.9 27.3 28.2 31.8 9.0
6.0 5.5 5.3 0.0 0.0 0.0 4.0 43.4
29.6 30.9 31.2 31.5 34.2 35.9 28.4 30.4
4. Discussion In the present study, we have investigated the Tm and reversibility of rmethuG-CSF by means of far-UV CD and compared the Tm obtained from the CD measurements with that from the IR study. According to the CD studies, the secondary structure of rmethuG-CSF is the most stable at pH 2.8, but its reversibility is the highest not at this pH but at pH 2.0. The result indicates that the net charge of rmethuG-CSF at pH 2.8 makes the conformation stable. RmethuG-CSF at pH 2.0 is more positively charged, resulting in the stronger protein–protein electrostatic repulsion. Once the conformation of rmethuG-CSF partially unfolds by heating, the electrostatic repulsion suppresses to form the aggregates. Balance between the effect of conformation stability and that of electrostatic repulsion determines the thermal stability of rmethuG-CSF. Border pH for this balance seems to be pH 2.8 under the conditions of our CD study. Recent studies of Chi et al. suggested that the aggregation of rmethuG-CSF is controlled by both conformation stability and colloidal stability and depends on the solution conditions [11]. Our conclusion is supported by the results of Chi et al. Generally, the protein concentration of more than 10 mg/ mL is required to obtain IR spectra. For far-UV CD analysis, the protein concentration of 0.1–1 mg/mL is usually required. Therefore, the region of protein concentration required for the measurements is different between IR and CD. In the thermal stability study of rmethuG-CSF, rmethuG-CSF is more easily influenced at pH 4.0 by the measurement conditions than at pH 2.5. The Tm of rmethuGCSF is easily affected by the measurement conditions such as protein concentration and heating rate as the reversibility is low. We cannot measure a CD spectrum of a protein with the concentration of 10 mg/mL, which is the suitable concentration for the IR measurement, because of high CD absorbance of the protein. Accordingly, we measured the CD spectra of rmethuG-CSF with the concentration of 5 mg/mL. When the CD spectra of rmethuG-CSF are measured for the protein concentration of 5 mg/mL and the heating rate of 0.2 8C/min, the Tm obtained from CD is almost identical with that from IR. The thermal denaturation of rmethuG-CSF is not fully reversible. Its reversibility is approximately 60% at pH 4.0.
While the amount of a-helix decreases from 70 to 35% at pH 4.0, those of b-structure and random coil increase, especially b-structure content becomes 30%. The b-structure appears to be intermolecular antiparallel b-structure, leading to the aggregation. On the other hand, the IR spectrum of rmethuG-CSF at 25 8C shows a dominant band at 1654 cm 1 in the amide I region, indicative of a-helix. As temperature increases, the intensity of the band at 1654 cm 1 decreases, and instead new bands appear at 1685 and 1618 cm 1 (Fig. 4). The bands indicate that intermolecular antiparallel b-structure is formed by aggregation [10]. The aggregation is produced as the consequence of the thermal denaturation of rmethuG-CSF. The b-structure observed by CD after heating seems to be the same as the intermolecular antiparallel b-structure detected by the IR spectra. This study demonstrates that the amount of bstructure increases as pH increases over a pH range of 2.0– 4.0 and at pH 5.0 the precipitates are formed. This means that b-structure is soluble in the pH range of 2.0–4.0, but at pH 5.0 b-structure is beyond solubility limit and the precipitation is formed. Recently, Chi et al. demonstrated that the increase in the repulsive interactions between protein molecules is effective in reducing the aggregation in a rmethuG-CSF solution at low pH [11]. Therefore, the decrease in the repulsive interaction between denaturated proteins seems to cause the precipitation at pH 5.0.
Fig. 4. IR spectra in the amide I region of rmethuG-CSF in a 20 mmol/L Na phosphate buffer at pD 4.0 at 25 and 80 8C. Solid line represents the IR spectrum at 25 8C, while dashed line represents the IR spectrum at 80 8C.
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K. Yamazaki et al. / Vibrational Spectroscopy 38 (2005) 33–38
When the thermal stability of a protein is assessed using Tm, it is important to take into account measurement conditions such as protein concentration and heating rate. Especially, when the unfolding reaction is at least partially irreversible, the difference in the measurement conditions has more influence on the evaluation of Tm.
5. Conclusion The thermal stability of rmethuG-CSF is highest at pH 2.8 while its reversibility is the highest at pH 2.0 in the pH range of 2.0–5.0 under the measurement conditions of CD. The conditions where the Tm is the highest are different from those where the reversibility is the highest. RmethuG-CSF at pH 4.0 is more influenced by the changes in the measurement conditions such as the protein concentration and heating rate, than that at pH 2.5 because the reversibility of rmethuG-CSF at pH 2.5 is higher than that at pH 4.0. When the thermal stability of protein is assessed by using Tm, measurement conditions are very important. One of the purposes of the present study is to compare the results for the Tm obtained by CD and IR spectra. It was found that when the CD spectra of rmethuG-CSF were measured for the heating rate of 0.2 8C/min and the protein concentration of 5 mg/mL, which is pretty high for usual CD measurements for protein solution while low for usual IR measurements,
the Tm obtained from CD is almost identical with that from IR.
Acknowledgement We thank Associate Professor F. Arisaka (Tokyo Institute of Technology) for valuable advice on the analysis of the CD data.
References [1] N.A. Nicola, Annu. Rev. Biochem. 58 (1989) 45. [2] G.D. Demetri, J.D. Griffin, Blood 78 (1991) 2791. [3] C.P. Hill, T.D. Osslund, D. Eisenberg, Proc. Natl. Acad. Sci. 90 (1993) 5167. [4] L.O. Narhi, W.C. Kenney, T. Arakawa, J. Protein Chem. 10 (1991) 359. [5] C.G. Kolvenbach, S. Elliott, R. Sachdev, T. Arakawa, L.O. Narhi, J. Protein Chem. 12 (1993) 229. [6] C.G. Kolvenbach, L.O. Narhi, J.S. Philo, T.S. Li, M. Zhang, T. Arakawa, J. Pept. Res. 50 (1997) 310. [7] C.N. Pace, K.L. Shaw, Proteins Struct. Funct. Genet. Suppl. 4 (2000) 1. [8] C.N. Pace, Trends Biotechnol. 8 (1990) 93. [9] J.T. Yang, C.S. Wu, H.M. Martinez, Methods Enzymol. 130 (1986) 208. [10] A.L. Fink, Folding Des. 3 (1998) R9. [11] E.Y. Chi, S. Krishnan, B.S. Kendrick, B.S. Chang, J.F. Carpenter, T.W. Randolph, Protein Sci. 12 (2003) 903.