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Supplementary Information
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Why Do Solution Additives Suppress the Heat-Induced Inactivation of Proteins?
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Inhibition of Chemical Modifications
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Shunsuke Tomita and Kentaro Shiraki*
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Institute of Applied Physics, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573,
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Japan,
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Supplementary Methods
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Determination of ammonia for RNase A
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Aliquots of 180 μl of heat-treated RNase A solutions were mixed with the following
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solutions: (i) 1160 μl of 200 mM Tris-HCl (pH 8.0), (ii) 120 μl of 100 mM 2-oxoglutaric acid (pH
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7.4), (iii) 50 μl of 5 mM NADH, 1% NaHCO3, (iv) 30 μl of 5 mM EDTA, and (v) 60 μl of 10
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U·ml-1 GDH, 200 mM Tris-HCl (pH 8.0). In the case of GlyAd, 45 μl of heat-treated RNase A
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solution was mixed with the following solutions: (i) 357.5 μl of 200 mM Tris-HCl (pH 8.0), (ii)
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37.5 μl of 2-oxoglutaric acid (pH 7.4), (iii) 30 μl of 5 mM NADH, 1% NaHCO3, (iv) 10 μl of 5 mM
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EDTA, and (v) 20 μl of 10 U·ml-1 GDH, 200 mM Tris-HCl (pH 8.0). After incubated at 4°C for 90
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min, the samples were centrifuged at 15000 × g for 20 min at 25°C. The decrease in NADH was
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estimated from the change in absorbance at 340 nm.
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Determination of ammonia for lysozyme
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Aliquots of 90 μl of heat-treated lysozyme solutions were mixed with the following
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solutions: (i) 327.5 μl of 200 mM Tris-HCl (pH 8.0), (ii) 37.5 μl of 100 mM 2-oxoglutaric acid (pH
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7.4), (iii) 15 μl of 5 mM NADH, 1% NaHCO3, (iv) 10 μl of 5 mM EDTA, and (v) 20 μl of 10
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U·ml-1 GDH, 200 mM Tris-HCl (pH 8.0). In the case of GlyAd, 45 μl of heat-treated RNase A
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solution was mixed with the following solutions: (i) 357.5 μl of 200 mM Tris-HCl (pH 8.0), (ii)
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37.5 μl of 2-oxoglutaric acid (pH 7.4), (iii) 30 μl of 5 mM NADH, 1% NaHCO3, (iv) 10 μl of 5 mM
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EDTA, and (v) 20 μl of 10 U·ml-1 GDH, 200 mM Tris-HCl (pH 8.0). After incubated at 4°C for 90
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min, the samples were centrifuged at 15000 × g for 20 min at 25°C. The decrease in NADH was
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estimated from the change in absorbance at 340 nm.
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Preparation of reduced RNase A
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Aliquots of 180 μl of heat-treated RNase A solutions were mixed with 360 μl of a solution
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containing 8 M Gdn and 150 mM PB (pH 8.0), and incubated at 25°C for 60 min. After incubation,
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4.5 μl of 1-octanol and 72 μl of 5.0 M NaBH4 were added to the samples to reduce the proteins.
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After incubation at 25°C for 75 min, 72 μl of 6 M HCl was gently added to the samples to acidify
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for decomposition of NaBH4. Then, 679.5 μl of 0.5 M Na-phosphate buffer (pH 10.0) and 72 μl of a
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solution of containing 5 mM DTNB and 150 mM Na-phosphate buffer (pH 8.0) were added to the
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samples. The samples were centrifuged at 15000 × g for 20 min at 25°C. Finally, the free sulfhydryl
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groups were estimated from the absorbance at 412 nm.
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Preparation of unreduced RNase A
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Aliquots of 180 μl of heat-treated RNase A solutions were mixed with 1035 μl of a solution
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containing 8 M Gdn and 150 mM PB (pH 8.0), and incubated at 25°C for 60 min. After incubation,
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135 μl of a solution of containing 5 mM DTNB and 150 mM Na-phosphate buffer (pH 8.0) were
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added to the samples. The samples were centrifuged at 15000 × g for 20 min at 25°C. Finally, the
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free sulfhydryl groups were estimated from the absorbance at 412 nm.
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Preparation of reduced lysozyme
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Lysozyme formed precipitates upon heating. As aggregate formation would complicate
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determination of the number of broken disulfide bonds, we roughly estimated the disulfide
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destruction using the following methods to avoid misinterpretation of the results. Aliquots of 200 μl
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of a solution containing 8 M Gdn and 150 mM PB (pH 8.0) were added to micro tubes containing
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100 μl of heat-treated samples, and incubated at 25°C for 60 min. After incubation, 2.5 μl of
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1-octanol and 40 μl of 5.0 M NaBH4 were added to the samples to reduce the proteins. After
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incubation at 25°C for 75 min, 40 μl of 6 M HCl was gently added to the samples. Then, 300 μl of
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each sample was mixed with 1000 μl of 500 mM Na-phosphate buffer (pH 10.0) and 100 μl of a
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solution containing 5 mM DTNB and 150 mM Na-phosphate buffer (pH 8.0). The samples were
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then centrifuged at 15000 × g for 20 min at 25°C. Finally, free sulfhydryl groups were estimated as
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described above.
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Preparation of unreduced lysozyme
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Aliquots of 200 μl of a solution containing 8 M Gdn and 150 mM PB (pH 8.0) were added
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to micro tubes containing 100 μl of heat-treated samples, and incubated at 25°C for 60 min. After
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incubation, 100 μl of a solution of containing 5 mM DTNB and 150 mM Na-phosphate buffer (pH
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8.0) were added to the samples. The samples were centrifuged at 15000 × g for 20 min at 25°C.
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Finally, the free sulfhydryl groups were estimated from the absorbance at 412 nm.
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Supplementary Figures
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Supplemental Figure S1. Time course of heat-induced inactivation on RNase A and lysozyme in
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the presence of additives in water (pH 7.0). Samples containing 1.0 mg·ml-1 RNase A (a) or
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lysozyme (b) in the presence of 200 mM additives were heated at 98°C for various periods. Key:
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None, closed circles; Arg, open circles; GlyAd, closed squares; Spd, open squares.
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Supplemental Figure S2. Far-UV CD spectra of RNase A and lysozyme in the presence of
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additives at different temperatures. Samples containing 0.2 mg·ml-1 RNase A (a) or lysozyme (b) in
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the absence (black) or presence of 200 mM NH4Cl (red), Spd (blue), or GlyAd (green) were
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incubated at 98°C (solid line) and 25°C (broken line).
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Supplemental Figure S3. Time course of heat-induced inactivation of RNase A and lysozyme
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with different concentrations or after refolded. Samples containing 1.0 mg·ml-1 (closed circles) or
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0.3 mg·ml-1 (open circles) RNase A (a) or lysozyme (b) in 50 mM Na-phosphate buffer (pH 7.0)
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were heated at 98°C for various periods. 1.0 mg·ml-1 heat-treated proteins refolded by Gdn were
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shown as closed squares.
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Supplemental Figure S4. Time course of heat-induced deamidation of RNase A and lysozyme.
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Samples containing 1.0 mg·ml-1 RNase A (a) or lysozyme (b) in 50 mM Na-phosphate buffer (pH
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7.5 (closed circles), 7.0 (open circles), or 6.5 (closed squares)) were heated at 98°C for various
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periods.
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Supplemental Figure S5. Time course of heat-induced β-elimination of RNase A and lysozyme.
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Samples containing 1.0 mg·ml-1 RNase A (a) or lysozyme (b) in 50 mM Na-phosphate buffer (pH
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7.5 (closed circles), 7.0 (open circles), or 6.5 (closed squares) were heated at 98°C for various
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periods.
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a
b
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Supplemental Figure S6. X-ray crystal structure of RNase A (a) and lysozyme (b) [PDB entry
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RNase A, 2AAS; lysozyme, 1HEL]. Asn-Gly sequenses (RNase A; Asn 67 and Gly 68, lysozyme;
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Asn 103 and Gly 104), red; the catalytic residues (RNase A; His 12 and His 119, lysozyme; Glu 35
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and Asp 52), blue.
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