Allosteric modulation of oxygen binding to the three human embryonic haemoglobins

Abstract

Plasmid based yeast expression systems have been developed for the high-level expression of the three human embryonic haemoglobins Gower I (zeta 2 epsilon 2), Gower II (alpha 2 epsilon 2) and Portland (zeta 2 gamma 2). Physiochemical characterization of the three product haemoglobins show them to be in the 'native' state. Oxygen-binding studies show that, under what are usually considered physiological conditions, each of the embryonic haemoglobins shows a high oxygen affinity, coupled to a high degree of co-operativity. Allosteric modulation of the oxygen-binding properties of the three haemoglobins in response to organic phosphates and protons has been investigated. The various responses exhibited by the three haemoglobins are rationalized in terms of their amino acid sequences.

Non‐synergistic interactions between strong allosteric effectors and human embryonic and adult haemoglobins

“…With regards to the embryonic proteins our data supports our earlier preliminary report of the differential responses of these proteins to D.P.G. which has established that a His 13143 -Ser difference in the ~, chain accounts for the low response of r [8]. In the case of Bzf binding we see that both the high and low affinity sites in ~2132 are preserved in the cz2~2 protein.…”

“…The three human embryonic haemoglobins were expressed in yeast using plasmid based expression systems as reported earlier [7]. Each protein was purified essentially as described in the literature [8] with the exception that final protein purification was achieved by chromatography on an immobilised Zn column. Oxygen binding curves were obtained using a Hemox analyser at 37 oc as previously described [9].…”

Sequence Divergence in the 3′ Untranslated Regions of Human ζ- and α-Globin mRNAs Mediates a Difference in Their Stabilities and Contributes to Efficient α-to-ζ Gene Developmental Switching

“…parisons and structural mapping studies suggest, however, that the major polypyrimidine tract is the most important feature determining ␣-globin mRNA stability and ␣-complex assembly (14,44). Thus, the effect of the single-base transversion on ␣-complex assembly was studied.…”

“…5A). The -globin 3ЈUTR was aligned to maximize homology to three pyrimidine-rich elements of the ␣-globin 3ЈUTR, which have been directly implicated in both ␣-globin mRNA stability and ␣-complex assembly (14,43,45). All three of the pyrimidine-rich tracts within the ␣-globin 3ЈUTR are conserved within the -globin 3ЈUTR.…”

“…Heparin (1 l of a 50-mg/ml stock) and loading dye were added, and the reaction was resolved on a 60:1 acrylamide-bis-acrylamide gel prerun at 110 V for 1 h in 1ϫ TBE. ␣-Complex assembly on ␣42, ␣42G, and 42 DNA oligomers was assessed by a similar method which omitted digestion with RNase (14). Pilot experiments (not shown) indicated that the ␣-complex assembles with equal affinity on sequence-identical RNA and DNA oligomers.…”