Globin Chain Analysis by Reversed Phase High Performance Liquid Chromatography: Recent Developments

Abstract

Reversed phase high performance liquid chromatography of globin chains is an important additional tool in the study of hemoglobin abnormalities. Using a technique modified from that of Leone et al.,[1] we report here the relative chromatographic behavior of about 200 different hemoglobin variants. This method provides an additional dimension in the presumptive characterization of hemoglobin variants. It was also found to be of special value for measuring the expression of neutral variants, such as thalassemic or unstable hemoglobins, and to identify neutral mutations associated with another variant, resulting in unusual hematological presentations.

Hb Barika [α42(C7)Tyr→His (α2)] Leads to an α+-Thalassemia-like Syndrome

“…0.7%, was observed in the C-window. Reversed phase HPLC analysis of the globin chains revealed an abnormal hydrophilic α chain, eluting with a retention time of 18 according to a relative scale where β and α elute at 10 and 20, respectively (7). Isopropanol stability test performed on the hemolysate was normal.…”

Detection of a Thalassemic α-Chain Variant (Hemoglobin Groene Hart) by Reversed-Phase Liquid Chromatography

“…Furthermore, when the mutant Hb cannot be isolated with conventional methods because of a low concentration or high hydrophobicity, we recommend RPLC analysis. Although one may choose among chromatographic techniques that have already been described (28,29 ), the new LC method we have described appears to be highly efficient for isolating hydrophobic molecules. Residues with hydrophobic side chains located in helices G and H are shown in orange; heme is presented in red.…”

Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography

“…By summing the change in the law of hydrophobicity (increase between 25% and 60% acetonitrile) according to previous similar works [12][13][14]21,23], the gradient was designed with a high percentage of solvent B at the beginning and ended with a lower percentage during several minutes within the determined scope of hydrophobicity. Our goal was obtaining a procedure for satisfactory separation of human globin chains.…”

“…Proper identification of an unknown Hb variant is important to determine if it is causing a clinical abnormality or is simply a polymorphism. Reversed phase highperformance liquid chromatography (RP-HPLC) could be useful for this approach [12][13][14]. Globin chain analysis is also useful for monitoring gene therapy and other related hematologic studies [15][16][17][18][19].…”