Impact Of Glycosylation On The Structure Of Human Hemoglobin

Glycated hemoglobin(Hemoglobin A1c,HbA1c),a typical glycosylation protein,is an important clinical biomarker for the early diagnosis.The level of HbA1c is closely related to the occurrence and development of diabetes complications,and is used as a criterion for the diagnosis.Glycosylation can lead to changes in the structure of Hb,causing a series of biochemical effects that ultimately alter the normal oxygen-transport functions of Hb.In order to reveal the functional disorders caused by glycosylation,and provide molecular-level information related to drug design for diabetes we have performed biochemical and biophysical studies on this system.In this thesis,HbA1c and non-glycosylated Hb(HbA0)were purified from normal blood as well as from diabetic patient samples;in addition,synthesis of glycosylated Hb(GHb)was achieved by the glycosylation of Hb with glucose in vitro.We utilized chromatography,spectroscopy,mass spectrometry,and X-ray crystallography to study the structural differences of HbA1c and HbA0 in nature,and to probe the factors affecting the degradation of GHb.First,two main factors which affect the degradation of GHb in vitro were obtained by orthogonal experiments.Second,HbA1c and HbA0 were isolated and purified from normal adults and diabetic patients,as well as GHb generated in vitro using ion exchange chromatography followed with boric acid affinity chromatography.The absorption spectrum and HPLC results of the eluents showed that the higher the degree of glycosylation,the stronger the ability to bind to the affinity column.Mass spectra of purified HbA1c showed a 162 Da mass increase in the beta chain compared to the counterpart of HbA0.Finally,Hb,HbA1c,and HbA0 samples were subjected to non-denaturing gel electrophoresis and spectroscopic characterization.Non-denaturing gel electrophoresis showed that GHb generated in vitro had complicated changes compared with normal Hb and diabetic Hbs.Molecular fluorescence spectroscopy revealed that HbA1c had higher tryptophan auto-fluorescence intensities compared to HbA0,suggesting a shift of tryptophan residues to the protein surface.Dynamic light scattering showed that HbA1c is easier to aggregate compared to Hb,and also Hbs purified from diabetic patients are easier to aggregate than those from normal blood.Thermal shift spectroscopy revealed that the isolation and purification processes resulted in a decrease in the melting temperature(Tm)of Hbs,and HbA1c had lower Tm value than HbA0.In addition,the HbA1c protein was subjected to crystallization screening to obtain crystals of HbA1c for X-ray diffraction.In conclusion,HbA and HbA1c isolated from cation exchange chromatography exhibit dimeric structures.The structural changes upon glycosylation of Hb are the major factors that cause protein aggregation.Moreover,the structural impact of the glycosylation of Hb in vitro is greater than that in vivo(diabetic patients).The glycosylation of Hb may be one of the important molecular mechanisms to cause diabetic complications.