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The ability of a number of heterocyclic metal chelators to deliver zinc into red cells, to release the liganded zinc to haemoglobin and thereby cause a left shift in the oxygen dissociation curve of intact red cells has been investigated. Incubation of neutrally charged zinc-pyrone and zinc-pyridin-2-one complexes with red cells led to the rapid accumulation of zinc within cells, whereas unliganded zinc in the form of zinc acetate, zinc chloride or zinc sulphate accumulated only slowly. The rate at which zinc was delivered to red cells by pyrone and pyridin-2-one ligands increased with increasing lipid solubility of the ligands. The uptake of zinc into both normal adult and sickle red cells was associated with a dose-dependent increase in the oxygen affinity of haemoglobin. The degree of left shift in the oxygen dissociation curve following the incubation of red cells with zinc-pyrone and -pyridin-2-one complexes suggests that these complexes may find application as agents to increase the oxygen affinity of haemoglobin in sickle cell disease and thereby decrease the probability of intravascular sickling at low tissue oxygen tensions. Ethylmaltol appears to be a particularly useful agent due to its known low toxicity.

The polymerisation of deoxyhaemoglobin S at 37 °C has been characterised by the supernatant concentration remaining after ultracentrifugation of the gel. Other haemoglobins, that are often found to occur with haemoglobin S, influence the polymerisation process. The effects of such haemoglobins have been quantitated in terms of a parameter, which estimates the difference in tendency to polymerise between a particular haemoglobin and haemoglobin S. The interpretation also assesses the participation of hybrid molecules present in mixtures of haemoglobins. The results suggest that, while each of the haemoglobins can form mixed polymers with haemoglobin S, haemoglobin F shows a greater inhibition of polymer formation than haemoglobin A, and haemoglobin A 2 has the maximum inhibitory effect. Hybrids show the same gelling potential as an equimolar mixture of each of these haemoglobins (A, F or A 2 ) with haemoglobin S. Haemoglobin C shows a large inhibition of polymerisation, intermediate to haemoglobins F and A 2 . However, hybrids of haemoglobins C and S behave like haemoglobin S and contribute preferentially to polymer formation. This behaviour could explain the severity of SC-disease relative to haemoglobin AS trait.

Desferrioxamine was given intravenously, at higher doses than previously reported, to counter the effects of transfusion-induced iron overload in four patients with beta thalassaemia major. In two of them retinal abnormalities developed, presenting with night blindness and field defects, which improved on withdrawal of the drug.

The amino acid compositions of the tryptic peptides of both chains (α Ami and β Ami ) of the normal major haemoglobin of Macaca irus (Hb-A mi ) have been analysed (excluding αT 12B and αT13). The most probable sequences of these chains have been constructed by comparison with the α- and β-chain sequences of human Hb-A and Macaca mulatta Hb-A. A minor component (α 2 Xmi β 2 Ami ) is found as a polymorphism in M. irus . Amino acid analysis of the soluble tryptic peptides of the α Xmi -chain has elucidated the positions of four amino acid differences between this chain and the α Ami -chain.

The reaction between p-chloromercuribenzoate (PCMB) and haemoglobin has been investigated to define the optimum conditions for dissociation to the subunits. The production of various molecular species has been followed by electrophoresis, gel filtration and ultracentrifugation. The reagent was applied to human haemoglobins A, S, C, Ja (Glasgow), A2, H and F, and also to the haemoglobins of dog, rabbit, horse and pig. Evidence is presented for the occurrence of haemoglobins without α-chains containing dissimilar β-chains, i.e. β2Yβ2X The behaviour of haemoglobins in the presence of PCMB was found to correlate qualitatively and quantitatively with the nature and number of their sulphydryl groups. It is shown that attachment of reagent at position β-93 causes initial dissociation of the tetramer to the dimer: α2β2 ag 2αβ. From the location of these reactive sulphydryls it is concluded that the initial dissociation is along the bc plane of the haemoglobin molecule with the formation of two α1β1 dimers. Further dissociation by PCMB at pH 6.0 only occurred with haemoglobins containing a second sulphydryl group on the β-chain (β-112). From the data it is concluded that substitution at both unreactive thiols β-112 and α-104 causes final dissociation to the monomeric species. The contribution of the various sulphydryl groups to dissociation was also indicated by the behaviour of alkylated haemoglobins when treated with PCMB. Evidence is presented that dissociation of haemoglobin to half-molecules in several media occurs across the same plane as that initially affected by PCMB. The connection between the sigmoid oxygen tension curve and dissociation is discussed.

Peripheral-blood mononuclear cells from an individual known to produce antibody to the rhesus D blood group were enriched for specific antibody-producing cells before transformation with Epstein-Barr virus. An anti-rhesus-D antibody-producing cell line (UCH D4) was obtained and cloned. Culture supernatants contained about 20 micrograms/ml of rhesus-D-specific IgG-class antibody. The antibody reactivity compared well with that of reagents used routinely for rhesus blood-grouping.

Plasma proteins glucosylated in vitro were injected repeatedly into 7 mice. A group of 4 was given multiple injections of non-glucosylated plasma proteins and another 10 were untreated. 5 of 7 mice given glucosylated proteins had glomerular-basement-membrane thickening on electron microscopical examination. This feature is characteristic of human and experimental diabetic renal disease. These results suggest that there may be a link between plasma-protein glucosylation and diabetic nephropathy.

Computer tomographic (CT) scans of the liver were obtained in six thalassaemic patients with iron overload confirmed by liver biopsy. Mean CT values for the liver in individual patients were linearly related to the iron content estimated by liver biopsy (correlation coefficient=0·995).

The appearance of two haemoglobin species in the recombination of canine haemoglobin with haemoglobin A has been confirmed by starch gel electrophoresis at two different pH's. The recombination of canine haemoglobin with haemoglobin H produced only the species with the higher anodic mobility. This species has therefore the composition α 2 A β 2 Can and the slower of the two species the composition α 2 A β 2 Can . A common species corresponding to α 2 A β 2 Can formed in recombination experiments with haemoglobins having abnormal β -chains and another common species corresponding to α 2 A β 2 Can in experiments with haemoglobins having abnormal α -chains. On the other hand the species α 2 A β 2 Can was not produced with the β -chain variants and the species α 2 A β 2 Can was absent in the experiments with the α -chain variants. Neither of these species was formed when canine haemoglobin was recombined with haemoglobin G/C. These results obtained with the human haemoglobin variants confirm the identification, made in the experiment using haemoglobin H, of the composition of the new species in the recombination of canine haemoglobin and haemoglobin A.

Procedures for the isolation of the β A - and γ F -chain haemoglobin subunits from normal adult and foetal haemoglobin respectively are described. Tryptic peptide analyses of both species show complete absence of α A -chain peptides and presence of the known β A - or γ F -chain peptides. Reaction of either species with isolated α A -chain subunits produces new species with the mobilities of normal adult or foetal haemoglobin respectively, at approximately the same rates as with naturally occurring Hb- β 4 F or Hb- γ 4 F . The β A -chain subunits have been characterized by their electrophoretic mobility in starch gel, rates of alkaline denaturation, absorption spectra and the γ F -chain subunits, also by their recombination with canine haemoglobin and sedimentation properties. They cannot be distinguished by any of these criteria from their naturally occurring counterparts, Hb- β 4 A and Hb- γ 4 F , respectively. It is concluded that the latter two species will form in vivo if more β A - and γ F -chains are produced than α A -chains.

Hb-A was absorbed on a oarboxymethyloellulose column. The first haemoglobin fraction eluted from this column by an acetate buffer pH 4·7 was heterogeneous. Electrophoretic analysis on starch block or in starch gel of the concentrated and neutralized fractions revealed three haemoglobin species. The species of intermediate mobility was present in greatest amount and consisted of Hb-A; that on the anode side of Hb-A was barely detectable. The species migrating nearest the cathode, amounting to 1 to 4% of the total pigment eluted from the column, had a mobility in starch gel at pH 8·7 less than that of Hb-G Br /C and an absorption spectrum and alkaline denaturation rate similar to those of Hb-A. Analysis of the tryptic peptides of this component showed that it contained only α A-polypeptide chains and it has therefore been named haemoglobin α A . Dissociation and recombination of Hb- α A with Hb-G Br /C produced one new species with the electrophoretic properties of Hb-C and another with the properties of α G Br -chains. Recombination with canine haemoglobin gave a single new haemoglobin zone migrating in the position of the species α 2 A β 2 Can and with Hb-I a single zone with the mobility of Hb-A. These results suggest that Hb- α A contains undegraded α A -chains whose configuration is such that they will, with the appropriate β -chains, reform typical tetramer haemoglobins. Hb- α A does not occur in vivo and the significance of this finding is briefly discussed.

The haemoglobin species α 2 A β 2 Can and α 2 F β 2 Can , α 2 Can and α 2 Can γ 2 F have been prepared and isolated from recombination mixtures of canine haemoglobin with haemoglobin A and foetal haemoglobin respectively. The species α 2 A β 2 Can and α 2 F β 2 Can cannot be distinguished by electrophoresis in starch or agar gels, rate of alkaline denaturation or position of their tryptophan fine structure bands. In the last two properties they resemble haemoglobin A. In contrast, the species α 2 Can γ 2 F and α 2 Can β 2 A differ in all these criteria in the same way as foetal haemoglobin differs from haemoglobin A. These results therefore suggest that the α -chains of haemoglobin A and foetal haemoglobin are identical and that the characteristic properties of foetal haemoglobin are determined by the γ 2 F subunits.