HX Procedure to Detect Freezing-Induced LDH

Denaturation in the Frozen State
HX labeling in the frozen state was conducted according to
the protocol shown in Fig. 1A. LDH stock solution following
dialysis and centrifugation was first diluted to desired
protein concentrations in H2O-based citrate buffer
(20 mM sodium citrate, 100 mM NaCl, pH 6.2). The
diluted LDH sample was then mixed at 1:9 volume ratio
with D2O-based citrate buffer with the same solution
composition and pH. The resultant LDH samples had
90% deuterium in solvent and desired final protein
concentrations of 0.02, 0.05 and 0.10 mg/ml. To minimize
H/D exchange in the solution phase before sample
freezing, the final mixing step of LDH solution and
deuterium buffer was carried out in the shortest time
possible for manual operation (less than 2 s), immediately
followed by flash freezing in liquid N2 for 5 min. Frozen
LDH samples were then incubated for the selected labeling
time at −10°C. This temperature was chosen because H/D
exchange took place within a reasonable experimental time
scale (e.g. hours to one day). Carrying out the labeling at a
temperature lower than the eutectic point of NaCl solution
(−21.2°C) would completely freeze the sample (i.e. no
remaining liquid in the frozen sample), thus leading to an
extremely slow H/D exchange rate. Thawing of the
deuterium-labeled frozen sample was achieved by adding
twice the sample volume of ice-cold 50 mM citrate buffer
(pH 2.5) containing 6 M guanidine HCl. Consequently,
back exchange was minimized by lowering the pH to ~2.8
during the thawing period. Further, the time for complete
thawing of the frozen sample was reduced to 2 min by the
high concentration of guanidine HCl.