My first indication of the appearance of nitrosative conditions in addition to nitrations during freezing with nitrite in sodium phosphate buffer was by freezing (–20°C) of metal-free solutions of albumin (25µM BSA) with or without 10 mM nitrite in sodium
5Universit¨atsklinikum Hamburg-Eppendorf, Hamburg, Germany, now at the Klinikum der Johannes Gutenberg-Universit¨at, Mainz, Germany.
or potassium phosphate buffer, followed by determination of S-nitrosation. Albumin is known to contain only one accessible cysteine (Cys-34) and this is susceptible to S -nitrosation.S-Nitrosation of BSA was determined by copper ion driven decomposition of S-nitrosothiols and detection of the resulting free •NO by DAN fluorescence. In samples which contained nitrite in sodium phosphate buffer and were frozen, 23.98± 5.93µM nitrosothiols were detected, whereas in all other samples the concentration was below 1µM. This nearly completeS-nitrosation of thiols from BSA could be prevented by blocking thiol groups with DTNB or iodoacetamide prior to freezing. In this case, nitrosation was decreased by more than 50 % (data not shown, see [261]).
0 5 10 15 20 25
+4 °C −20 °C
NO [µM]
Figure 5.15: S-Nitrosation of albumin during freezing in sodium phosphate buffer. Samples containing BSA and 1 mM sodium nitrite in 100 mM sodium phosphate buffer (pH 7.4) were stored over night at 4°C or frozen at –20°C prior to determination of S-nitrosation. S-nitrosation was measured with the oxyHb assay by releasing•NO fromS-nitrosothiols with Cu2+. Data are mean values ±SD; n = 4.
Determination of S-nitrosation by Cu2+ and DAN turned out to be not a reliable method, perhaps due to the fact that small traces of nitrite will disturb the assay.
Therefore, a similar experiment was done, whereas quantification of nitrosation was performed with a modified oxyHb assay (Fig. 5.15). Although the addition of
two-charged metal ions results in the formation of an insoluble precipitation of potassium salts, which in turn disturbs the optical method, this method provides reliable results.
A high level of nitrosation was detected after freezing of the samples, but the • NO-signal was lower as in the first experiment; this could be due to the lower concentration of nitrite.
5.5.2 N-Nitrosation of 2,3-Diaminonaphthalene
0 20 40 60 80 100 120 140 160 180 200
10 µM nitrite w/o nitrite 10 µM nitrite w/o nitrite
NAT fluorescence [RLU]
+4 °C −20 °C
NaPi buffer KPi buffer
Figure 5.16: N-Nitrosation of DAN during freezing in sodium phosphate buffer.Upon storage of samples containing sodium phosphate (100 mM, pH 7.4), nitrite (10µM) and DAN (30µM) for 2.5 h at −20°C, a fluores-cence signal was observed, whereas in the absence of nitrite or in samples containing potassium instead of sodium phosphate no significant forma-tion of NAT was detectable. In addiforma-tion, only background fluorescence was observed in samples which were kept at 4°C for the same time. Data are mean values± SD; n= 3.
In a third experimentN-nitrosation was directly investigated using DAN as a substrate for nitrosation during freezing (Fig. 5.16). This direct nitrosation of DAN results in higher sensitivity and lower error values. Therefore, it was possible to investigate the effects of lower concentrations of nitrite and the used concentration of 10µM matches
the physiological cytosolic concentration. Upon storage of samples containing sodium phosphate, nitrite and DAN at –20°C a high fluorescence signal of NAT was observed, whereas in the absence of nitrite and/or sodium or in samples which were kept at +4°C only basal fluorescence was detected. These results show that in addition to S -nitrosation also N-nitrosation is likely to occur during freezing of samples in sodium phosphate buffer and the presence of micromolar concentrations of nitrite is sufficient for this.
Physiological levels of nitrite in cell culture media are around 0.5µM and even 10µM in the cytosol but can reach up to 80µM in media containing cells or tissue with induced NOS. In our experiments 1µM nitrite was sufficient to see covalent protein modifications [261, 262]. Depending on the temperature, we observed different levels of nitration and nitrosation. Storage at –20°C results in the highest yield of sample modification, at –70°C the effects were significantly lower and even lower during freezing in liquid nitrogen (–196°C). But longer storage at these temperatures does not result in higher yields of nitration and nitrosation. Therefore, we determined the velocity of freezing as a key factor, in addition to the presence of sodium phosphate and nitrite. Slow freezing respectively freezing at higher temperatures results in prolongation of the conditions were a solid and a liquid phase is present in the samples.
That freezing of sodium phosphate buffered solutions leads to unexpected results was first demonstrated byGomezet al.[263]. During the freezing process, the more acidic H2PO−4 anion will accumulate in the liquid phase, whereas sodium salts of the alkaline anions HPO2−4 and PO3−4 precipitate or co-crystallize with the aqueous phase. This phenomenon will cause a decline in the pH of the liquid phase, even reaching values between 3 and 4. The slower the freezing process, the more distinct the shift in pH will be, in accordance with the fact that sodium phosphate buffer already crystallizes during storage at 4°C.
The chemistry behind the nitrosation under such conditions is quite obvious in principle but more complex in detail. During freezing of Na2HPO4/NaH2PO4solutions the pH drops below the pKa of nitrous acid and hence the acidic solution becomes a nitrosating medium, possibly via N2O3 formation:
NO−2 +H
−−−−−+*
)−−−−− HNO2 (2)
2HNO2 −−−−−→−H2O N2O3 (3)
RSH + N2O3 −−−−−→ RSNO + NO−2 + H+ (4)
The acidification of nitrite solutions represents a common mechanism for synthesis of S-nitrosated cysteine, glutathione or albumin [227, 228].
In addition to nitration of proteins during freezing of biological samples, as was investigated by Andreas Daiber, I discovered nitrosation to play a significant role. These nitrosations occur as S-nitrosations of biological cysteine residues, as are present in GSH or proteins and also as N-nitrosation, as seen with the artificial target DAN. The model of nitration due to a decrease in pH was confirmed by the observation of nitrosation, which is an expected reaction under acidic conditions.
The low levels of nitrite which are necessary to yield high levels of nitrosation show that freezing of samples is likely to be a common source of artifacts and since nitrosation is under investigation as a common regulatory mechanism comparable to phosphorylation/dephosphorylation, one has to take care to exclude these artificial nitrosations (and nitrations). In conclusion, an appropriate buffer composition and fast freezing in liquid nitrogen are necessary to avoid these artifacts. Whereas this does not play a rolein vivo, the newly discovered mechanism of nitrosation is of significant importance for the interpretation of collected data from biological samples.