R. W. Carrell

A Simple Method for the Detection of Unstable Haemoglobins

Summary. A simple test is described for the detection of unstable haemoglobins by the addition of haemolysate to an isopropanol/buffer solution at 37°C. Over 200 normal haemolysates have been tested giving no precipitation up to 30 min incubation whilst six samples containing an unstable haemoglobin gave a precipitate within 5 min, which was flocculent by 20 min. As well as being a simple and effective screening test, the procedure is suitable for the purification of unstable haemoglobins for further studies but suffers from limitations, probably in common with the heat precipitation method, in the quantitation of the abnormal haemoglobin. Its use as a research tool is illustrated by comparative measurements of the stabilities of various forms of haemoglobin. These show deoxyhaemoglobin to be considerably more stable than oxyhaemoglobin thus supporting suggested interchain bondings in deoxyhaemoglobin.

α1-Antitrypsin and the serpins: variation and countervariation

n Abstractn n n αn 1-Antitrypsin is a plasma protein which protects elastic tissue from proteolytic attack. Consequently, genetic deficiency, or the oxidation of its reactive centre in cigarette smokers can result in the degenerative lung disease emphysema. Structural studies explain the mechanisms involved and have also drawn attention to a new family of serine proteinase inhibitors. The specificity of each of these inhibitors is primarily dependent on a single amino acid at its reactive centre. Site-directed mutagenesis is enabling the production of specifically designed inhibitors for therapeutic use, including an improved replacement for αn 1-antitrypsin deficiency.n n

Activated oxygen and haemolysis.

Oxidation is a major contributor to the degenerative processes that lead to ageing and cellular breakdown (Dormandy, 1969). This must be particularly true of the red cell which is at increased risk due to both its exposure to high concentrations of oxygen and its inability to replace damaged components by resynthesis. As a consequence, the red cell devotes much of its metabolic activity to reductive processes that combat thc threat of oxidation. When these reductive processes are deficient, or are overwhelmed, oxidation of cellular constituents will occur with consequent haemolysis. This is the basis of a characteristic group of diseases, the oxidative liaemolytic anaemias (Gordon-Smith & White, 1974). Although the general sequence of events leading to haemolysis in this disease group has been studied for some time, it is only recently that the precise nature of the oxidative process has begun to be understood. The direct oxidative threat is not posed by oxygen itself but rather by a series of high energy derivatives of oxygen. Thesc derivatives are easily interconvertible and it is experimentally difficult to pinpoint which of them is neccssarily responsible for the effects observed in the red cell. For this reason, it is useful to refer to the derivatives collectively as activated oxygen. This term simplifies discussion of what is a complex and as yet incompletely understood area.

α1-antitrypsin microheterogeneity: Isolation and physiological significance of isoforms

alpha 1-Antitrypsin has a microheterogeneity evident on isoelectric focusing as three major and several minor bands. We have identified the carbohydrate structures of the major bands; band 6 (isoform I) has three bi-antennary sidechains, band 4 (isoform II) has two bi- and one tri-antennary and band 2 (isoform III) has one bi- and two tri-antennary sidechains. The identity of the isoforms with the bands permitted their measurement in plasma by photometric scanning of the electrofocused gels. In healthy controls the levels of isoforms I, II and III were relatively constant and in the proportions of 5, 4 and 1, respectively. A marked change occurred during inflammation and oestrogen stress with isoforms II and III accounting for most of the increase in alpha 1-antitrypsin. One possible consequence of the changed proportions was shown to be the increased catabolism of the partially desialylated tri-antennary isoforms compared to that of the predominant bi-antennary form of the healthy individual.

Studies of Hemoglobin Denaturation and Heinz Body Formation in the Unstable Hemoglobins

The sequential changes that occur during the precipitation on mild heating of the unstable hemoglobins, Hb Christchurch, Hb Sydney, Hb Köln, and Hb A, were examined with particular attention to the possibility of an accompanying oxidative process. Hb Christchurch, Hb Sydney, and Hb A precipitated with equal amounts of alpha- and beta-chains and full heme complement. Hb Köln, however, was one-half hemedepleted and showed a slight excess of precipitated beta-chains. In all cases the spectrum of the precipitated material was typical of a hemichrome. There was no evidence that sulfhydryl oxidation contributed to the precipitation process. Reduced glutathione was unable to protect the hemoglobin against precipitation, and mixed disulfide formation between the precipitating hemoglobin and glutathione was insignificant, even in the presence of excess glutathione. No blockade of beta93 cysteines could be demonstrated in the unstable hemoglobins. Precipitation of oxyhemoglobin and carboxyhemoglobin in all cases gave nonspecific oxidation of approximately two of the six hemoglobin sulfhydryl groups to give intra- and intermolecular disulfide bonds. Single alpha- and beta-chains, plus polymers of up to five or six chains linked by disulfide bridges, were demonstrated by polyacrylamide gel electrophoresis. This disulfide oxidation was not observed with deoxy- or methemoglobin and did not appear to influence the rate of precipitation. These findings fit the theoretical prediction that autoxidation of oxy- and carboxyhemoglobin is accompanied by formation of a free radical, with the reactions of this free radical being confined intramolecularly.Together, these results are in keeping with predictions based on the known structural abnormalities of the unstable hemoglobins, all of which result in greater molecular flexibility. Our findings support the conclusion that the usual precipitating event is altered bonding at the heme to give the formation of hemichromes. There is no evidence of an accompanying oxidative process that could pose a threat to the integrity of the red cell.

Human α1-antitrypsin: carbohydrate attachment and sequence homology

Human err-antitrypsin (or-AT) has 3 carbohydrate sidechains [ 11. The attachment point of one of these sidechains has been determined in conjunction with the amino acid sequence of the C-terminal third of the molecule [2]. We now provide further sequence data which define the attachment points of the other 2 carbohydrate sidechains. These data also show the extent of the sequence homology with antithrombinIII and ovalbumin, and provide support for a single reactive centre situated near the C-terminus. tryptic digestion in 2 M guanidine hydrochloride in 0.1 M NI&HCOa buffer, pH 8.0 at 37’C for 15 h with an enzyme to substrate ratio of 1: 10.

Carboxy terminal fragment of human α-1-antitrypsin from hydroxylamine clevage: Homology with antithrombin III

Abstract Human α-1-antitrypsin (AT) was reacted with hydroxylamine at pH 9.0 giving cleavage at an Asn-Gly bond. A fragment of molecular weight 8,500 was released and this was isolated and sequenced. The fragment had the same carboxy terminal amino acid sequence as intact AT. The 80 residue polypeptide contained the Z variant mutation site and a portion of sequence identical to that found by others for the reactive site, inferring the presence in AT of two active sites. This sequence combined with prviously published work gives a continuous sequence of 152 amino acid residues from the carboxy terminal end of the AT molecule, including the mutation site of the S variant. The sequence shows strong homology with human antithrombin III.

Circulating proalbumin associated with a variant proteinase inhibitor

The unique finding of normal proalbumin in human plasma provides an insight into the mechanism of propeptide cleavage. Proalbumin, present as 1-5% of the total albumin, was found in a boy whose prime problem was the presence of a mutant proteinase inhibitor, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) [2]. The inferred structure of human proalbumin was confirmed as Arg-Gly-Val-Phe-Arg-Arg-Alb. On incubation with various enzymes (trypsin, tryptase, thrombin, chymotrypsin, chymase and cathepsin B), only trypsin was capable of converting proalbumin to albumin. There was no conversion when proalbumin was incubated with whole blood, plasma or serum. However, intravenous injection of proalbumin into a rat resulted in complete conversion to albumin, the half-life of this process being 6 h. We conclude that propeptide cleavage is dependent on a serine proteinase which is inhibited intracellularly, by the mutant inhibitor, and that all the albumin in the boy was secreted as proalbumin, but was subjected to a separate cleavage process after export from the hepatocyte.

The abnormality of the S variant of human α-1-antitrypsin

Abstract The tryptic peptide map of the S variant of α -1-antitrypsin differs in only one peptide from that of the normal M map. The amino acid sequence of this fifteen residue peptide is reported, the S peptide having a valine in position 5 compared to a glutamic acid in the same position in the M peptide. The abnormality has been confirmed in two unrelated donors with the SS phenotype.

The attachment of Heinz bodies to the red cell membrane.

Summary. The binding between Heinz bodies or heat‐precipitated haemoglobin and the red cell membrane was investigated. Ghosts containing Heinz bodies prepared from red cells containing Hb Christchurch and ghosts bound to heat‐precipitated Hb A were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Using this technique, which separated the membrane and Heinz body proteins according to size, no protein bands corresponding to derivatives of haemoglobin covalently bound to a ghost protein were detected. Binding between red cell ghosts and heat‐precipitated Hb A was also studied by density gradient ultracentrifugation, which separated ghosts bound to haemoglobin from pure ghosts and pure heat precipitate. Sulphydryl reducing and blocking agents were found to have no effect on the attachment of the insoluble haemoglobin to the ghosts, and reduction of electrostatic interactions in 2 m‐NaCl was also ineffective.