Cryoprotective Ability of Betaine-Type Metabolite Analogs During Freezing Denaturation

Supplementary Data

Cryoprotective ability of betaine-type metabolite analogs during freezing denaturation of enzymes

Yuichi Nakagawa,a Masahiro Sotab and Kazuya Koumoto*a

a Department of Nanobiochemistry, Frontiers of Innovative Research in Science and Technology(FIRST), Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.

b Research & Development Center, Nagase & Co., Ltd., 2-2-3 Murotani, Nishi-ku, Kobe, Hyogo 651-2241, Japan

Contents

1.Plot of remaining activities of α-GLU in the absence of cryoprotectants as a function of freeze–thaw cycles

2.Plots of the remaining activities of α-GLU as a function of concentration of analogs 7–14 after 12 freeze–thaw cycles

3.Comparison of the remaining activities of various enzymes in the absence and presence of analog 4 as a function of freeze–thaw cycles

4.Comparison of CD spectra of α-GLU in the absence and presence of analog 4 before and after 12 freeze–thaw cycles

Supplementary Fig. 1 Plot of remaining activity of α-GLU in the enzyme stock solution as a function of freeze–thaw cycles. Cryopreservation conditions: [α-GLU] = 25 µg/ml, [phosphate buffer (pH 7.0)] = 100 mM, freeze–thaw cycles (fast freezing) = 0–50 times.Error bars represent the standard deviation of five measurements.

Supplementary Fig. 2 Plots of remaining activities of α-GLU in the stock solution as a function of concentration of analogs 7–14 after 12 freeze–thaw cycles. Cryopreservation conditions: [α-GLU] = 25 µg/ml, [phosphate buffer (pH 7.0)] = 100 mM, [analogs] = 0–1000 mM, freeze–thaw cycles(fast freezing) = 12 times.Error bars represent the standard deviation of five measurements.

Supplementary Fig. 3 Plots of remaining activities of LDH, ALP, HRP, β-GLU, and SUL in the enzyme stock solutions as a function of freeze–thaw cycles (fast freezing) (0–50 times). Cryopreservation conditions: α-GLU: [α-GLU] = 25 µg/ml, [phosphate buffer (pH 7.0)] = 100 mM, [analog 4] = 300 mM,β-GLU: [β-GLU] = 50 µg/ml, [phosphate buffer (pH7.0)] = 100 mM, [analog 4] = 300 mM, ALP: [ALP] = 50 µg/ml, [Tris buffer (pH 7.5)] = 100 mM, [analog 4] = 300 mM, SUL: [SUL] = 625 µg/ml, [phosphate buffer (pH 7.0)] = 100 mM, [analog 4] = 300 mM, LDH: [LDH] = 50 µg/ml, [Tris buffer (pH 7.5)] = 80 mM, [analog 4] = 300 mM, HRP: [HRP] = 1.0 µg/ml, [citric acid buffer (pH 4.2)] = 10 mM, [analog 4] = 300 mM.Error bars represent the standard deviation of five measurements.

Supplementary Fig. 4 Comparison of circular dichroisim (CD) spectra of α-GLU in the (a) absence and (b) presence of analog 4 before (solid line) and after 12 freeze–thaw cycles (broken line). Conditions: [α-GLU] = 0.5 mg/ml, [phosphate buffer (pH 7.0)] = 100 mM, [analog 4] = 0 or 300 mM at 30°C, freeze–thaw cycles(fast freezing) = 0 (solid line) or 12 times (broken line).

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