Asunto(s)
ADN/química , Técnicas Genéticas , Hemoglobinas/biosíntesis , Hemoglobinas/síntesis química , Mutación , Acetona/farmacología , Animales , Aspirina/análogos & derivados , Aspirina/farmacología , Endopeptidasas/farmacología , Globinas/síntesis química , Globinas/química , Hemoglobina A/síntesis química , Hemoglobina A/química , Hemoglobinas/química , Hemoglobinas/genética , Humanos , Metahemoglobina/análogos & derivados , Metahemoglobina/farmacología , Urea/farmacologíaRESUMEN
An in vitro system that is independent of any haemodynamic influence and other physiological controls was designed to study the formation of carbamylated and glycosylated haemoglobin, by the use of cyanate and glucose respectively, incubated with whole-blood from normal healthy subjects. In this system the formation of glycosylated haemoglobin is more favourable than that of carbamylated haemoglobin. The effect of glucose in reducing the formation of carbamylated haemoglobin is greater than the effect of cyanate on the formation of glycoslyated haemoglobin. There is a strong correlation coefficient (r = 0.9, P < 0.005) for the relationship between the formation of carbamylated and glycosylated haemoglobin in this system.
Asunto(s)
Cianatos , Glucosa , Hemoglobina Glucada/síntesis química , Hemoglobina A/análogos & derivados , Hemoglobinas , Hemoglobina A/síntesis química , Humanos , Técnicas In VitroRESUMEN
The optimal conditions for the semisynthesis of alpha-globin through Staphylococcus aureus V8 protease condensation of a synthetic fragment (alpha 1-30) with the complementary apo fragment (alpha 31-141) in the presence of structure-inducing organic cosolvents and the reconstitution of the functional tetramer from semisynthetic alpha-globin have been investigated. The protease-catalyzed ligation of the complementary apo fragments alpha 1-30 and alpha 31-141 proceeds with very high selectivity at pH 6.0 and 4 degrees C in the presence of 1-propanol as the organic cosolvent. A 30% 1-propanol solution was optimal for the semisynthetic reaction, and the synthetic reaction attained an equilibrium (approximately 50%) in 72 h. The synthetic reaction proceeds smoothly over a wide pH range (pH 5-8). Besides, the semisynthetic system is flexible, and it also proceeded well if trifluoroethanol or 2-propanol was used instead of 1-propanol. However, glycerol, a versatile organic cosolvent used in all other proteosynthetic reactions reported in the literature, was not very efficient as an organic cosolvent in the present synthetic reaction. The semisynthetic alpha-globin prepared with 1-propanol as the organic cosolvent has been reconstituted into HbA. The semisynthetic HbA was then purified by CM-cellulose chromatography. The semisynthetic HbA is indistinguishable from native HbA, in terms of its structural and functional properties. The semisynthetic approach provides the flexibility in protein engineering studies for the incorporation of spectroscopic labels (13C- and/or 15N-labeled amino acids), noncoded amino acids, or unnatural bond functionalities, which at present is not possible with genetic approaches.