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. 2012 Aug 28:5:16.
doi: 10.1186/2046-1682-5-16.

Effects of spermine NONOate and ATP on the thermal stability of hemoglobin

Rasha Bassam  1 , Juergen Hescheler, Ayseguel Temiz-Artmann, Gerhard M Artmann, Ilya Digel
Affiliations

Effects of spermine NONOate and ATP on the thermal stability of hemoglobin

Rasha Bassam et al. BMC Biophys. .

Abstract

Background: Minor changes in protein structure induced by small organic and inorganic molecules can result in significant metabolic effects. The effects can be even more profound if the molecular players are chemically active and present in the cell in considerable amounts. The aim of our study was to investigate effects of a nitric oxide donor (spermine NONOate), ATP and sodium/potassium environment on the dynamics of thermal unfolding of human hemoglobin (Hb). The effect of these molecules was examined by means of circular dichroism spectrometry (CD) in the temperature range between 25°C and 70°C. The alpha-helical content of buffered hemoglobin samples (0.1 mg/ml) was estimated via ellipticity change measurements at a heating rate of 1°C/min.

Results: Major results were: 1) spermine NONOate persistently decreased the hemoglobin unfolding temperature Tuirrespectively of the Na + /K + environment, 2) ATP instead increased the unfolding temperature by 3°C in both sodium-based and potassium-based buffers and 3) mutual effects of ATP and NO were strongly influenced by particular buffer ionic compositions. Moreover, the presence of potassium facilitated a partial unfolding of alpha-helical structures even at room temperature.

Conclusion: The obtained data might shed more light on molecular mechanisms and biophysics involved in the regulation of protein activity by small solutes in the cell.

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Figures

Figure 1
Figure 1
Best-fit tangential line method for protein unfolding temperature calculation. Calculation of the unfolding temperature (Tu) using the best-fit tangential lines method, which is the intersection point between the first gentle line slope and the second steep slope.
Figure 2
Figure 2
Influence of ATP and spermine NONOate on unfolding of Hb samples prepared in Na-buffer. Influence of ATP and spermine NONOate on unfolding of Hb samples prepared in Na-buffer. a) control groups vs. NONOate groups; b) ATP groups vs. ATP+NONOate groups; c) control groups vs. ATP groups; d) NONOate groups vs. ATP+NONOate groups.
Figure 3
Figure 3
Influence of ATP and spermine NONOate on unfolding of Hb samples prepared in K-buffer. Influence of ATP and spermine NONOate on unfolding of Hb samples prepared in K-buffer. a) control groups vs. NONOate groups; b) ATP groups vs. ATP+NONOate groups; c) control groups vs. ATP groups; d) NONOate groups vs. ATP+NONOate groups.
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