1. Introduction
High performance liquid chromatography (HPLC) is an
advanced chromatographic method widely used for
detection of human hemoglobin (hHb) variants. Former
investigations by G. M. Artmann’s group using micropipette
aspiration technique, circular dichroism and dynamic light
scattering showed the existence of Hb structural transition
point at around 37 ᴼC. According to our current
understanding of the mechanisms of this transition, it is
related to a non-linear change of the surface
hydrophobicity of Hb with temperature.
In this study, the method of
reversed-phase HPLC was used to
detect changes in overall
hydrophobicity of Hb-samples by
analysis of their retention times.
The influence of different mobile
phase compositions on hemoglobin
structure was previously tested by
recording the absorbance spectra
with the Jasco V-550
spectrophotometer and Jasco HPLC
using a C4 column. The retention
time data were collected for seven
temperature steps between 25 °C to
40 °C in eleven repeated
independent measurements.
Fig .1: Average retention times for peak 1 plotted against temperature
Fig .2: Average retention times for peak 2 plotted against temperature Fig .3: Average retention times for peak 3 plotted against temperature
New Generation of Hemoglobin Structural Transition
Studies: RP-HPLC
Rasha Bassam1, 2, Mirella Fiebes1, Dariusz Porst1, Aysegül Temiz Artmann1, Gerhard M. Artmann1,
Andre Hütz3, Peter Kayser1, Ilya Digel1
1 Institute for Bioengineering, FH Aachen University of Applied Sciences, D-52428 Jülich
2 Institute of Neurophysiology, University of Cologne, Robert-Koch-Str. 39, 50931 Cologne
3 Jasco Deutschland GmbH
bassam@fh-aachen.de
Although more measurements need to be done to provide a robust statistical proof, the results showed a mostly linear
decrease of retention times with increasing temperature except a small fluctuation at 37 °C. Thus, our pilot RP-HPLC
measurements seemed to support the current Hb-transition model.
FH Aachen, Campus Jülich, Heinrich-Mußmann-Str. 1, D-52428 Jülich
Tel.: +49(0)241/6009-53139, Fax.: +49(0)241/6009-53273, www.zbp.fh-aachen.de
N=11
Fig .4: Structural transition of hemoglobin molecule at 37 ᵒC