Structure determination of porcine haemoglobin

Abstract

To investigate a potential candidate material for making artificial red blood cells to supplement blood transfusion, the X-ray structure of porcine haemoglobin at 1.8 A resolution was determined as part of research towards synthesizing human blood. Porcine haemoglobin was crystallized by the vapor-diffusion method, producing crystals of dimensions 0.3-0.5 mm after successive seeding. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 68.10, b = 72.27, c = 114.85 A. The initial phase was determined by the molecular-replacement method, using human oxyhaemoglobin as a model. The final R factor was 21.1% for 36 820 reflections after validation of 574 water molecules. The r.m.s. deviations of bond lengths, angles, torsion angles and improper angles from their ideal values are 0.017 A, 3.0, 20.6 and 1.8 degrees, respectively. The average B factor is 33.63 A(2) for the haemoglobin molecule and 50.53 A(2) for the water molecules. The structure could be superimposed on a 2.8 A resolution structure with an r.m.s. difference of 0.59 A in main-chain atomic positions and 1. 27 A in side-chain atomic positions. Porcine and human haemoglobins are compared. A tentative model for artificial blood is proposed based on the complementarity relationship of the surface charges between haemoglobin and the surrounding cell membrane.

Examination of the temporal variation of peptide content in decomposition fluid under controlled conditions using pigs as human substitutes

“…Peptide sequences were mapped against reference sequences of haemoglobin subunit alpha (XP_020942625) [30] , haemoglobin subunit beta (NP_001138313) [31], beta-enolase (NP_001037992) [32], creatine-kinase (NP_00112342) [33] and pyruvate kinase (XP_001929104) [34] to identify breakdown trends on each sample day. Consistent breakdown patterns were identified for Cadavers 1, 2 and 4 for both haemoglobin subunit alpha and beta, however, breakdown patterns for beta-enolase, creatine kinase and pyruvate kinase showed inconsistencies between the three cadavers.…”

“…greater potential exposure to proteolytic enzymes at these particular regions, likely increasing their susceptibility to proteolysis [30].…”

“…Peptides found to be most abundant were derived from residues 15-34 and 45-63 for each cadaver. As for haemoglobin subunit alpha, the nominated ranges of residues are potentially more exposed and therefore may be more susceptible to hydrolysis by proteolytic enzymes [30].…”

Heme iron state in various oxyhemoglobins probed using Mössbauer spectroscopy with a high velocity resolution

“…These hemoglobins have structural and functional differences (see Novy et al 1973;Sinet et al 1982, Rovida et al 1983Shaanan 1983;Uchida et al 1998;Lu et al 2000). The differences in amino acid composition in the a-and b-subunits in these hemoglobins are shown in Table 1.…”

Measurements of accurate x-ray scattering data of protein solutions using small stationary sample cells

“…The best crystals, up to 1.0 mm in their maximum dimension, were obtained from the Index F3 conditions ͑5% v/v Tacsimate pH7.0; 0.1M HEPES pH7.0 buffer; precipitant: polyethylene glycol monomethyl ether 5000͒. Crystallization of porcine hemoglobin ͑pHb͒ was carried out using the vapor-diffusion method under crystallization conditions similar to those described previously: 22 pHb was dissolved in distilled water to a concentration of 60-80 mg/ml, and a 2.8M phosphate solution, pH6.5-7.5 ͑made by mixing K 2 HPO 4 and NaH 2 PO 4 ͒, was used as the precipitant. The best pHb crystal diffracted to 1.9 Å resolution.…”