The Microheterogeneity of Human Haptoglobin and Its Complex with Hemoglobin

Abstract

and PRZYBYI.SKA, M. The microheterogeneity of human haptoglobin and its complex with hemoglobin. Can. J. Biochem. 51,597-685 ( 1973 ).Human haptoglobin, type 1s-IS, isolated from plasma and from ascites fluid was subjected to isoelectric focusing in polyacrylamide gel and its complex with horse cyaiaomethemoglobin was analyzed by isoelectric focusing in a column using a sucrose density gradient. Both methods revealed microheterogeneity. Similar patterns were obtained consisting of five to eight strong bands with a symmetrical distribution giving the highest yield in the center. An investigation of the nature of these bands was carried out and it was found that the sialic acid content varied in the different components. Experiments were undertaken to show that the multiple bands were not due to the binding of Ampholine to the protein and that this heterogeneity preexisted in haptoglobin prior to its isolation. No observable difference in heterogeneity was observed between samples of haptoglobin isolated by different methods and for complexes obtained by adding hemoglobin to haptoglobin and haptoglobin to hemoglobin.The unfractionated complex and the separated complex components were crystallized. YANG, N. J., et PRZYBYESKA, M. The microheterogeneity of human haptoglobin and its complex with hemoglobin. Can. J. Biochem. 51, 597-605 ( 1973 ). E'haptoglobine humaine de type IS-IS, isolCe du plasma et du liquide des ascites, est soumise la focalisation isoClectrique dans un gel de polyacrylamide et son complexe avec la cyano-mCthCmoglobine de cheval est analyst5 par focalisation isoCleetrique sur colonne, en prtsence d'un gradient de densit6 de saccharose. Les deux mCthodes rCvklent la microhCtCrog6nCitC. Les profils obtenus sont semblables et ils sont form& de einq B huit bandes importantes, distribuies de faqon symttrique, donnant un rendement plus ClevC dans le centre. La recherche de la nature de ces bandes nous a montrC que la teneur en acide sialique varie dans les diffCrents composCs. Des expCriences ont dCmontrC que les bandes multiples ne sont pas dues B la liaison de 1'Ampholine B la protCine et que cette httCrogCntit6 existait dCjB dans l'haptoglobine avant son isolation. Nsus n'avons observt aucune diECrence dans IVhCtCrogCnCitC entre les Cchantillons d'haptoglobine isolCe par diffCrentes mCthsdes et pour les complexes obtenus par addition d'hemoglobine B l'haptoglobine et d'haptoglobine h I'hCmoglobine. Le complexe non fractionnC et les constituants sCparCs du complexe sont cristallisCs. [Traduit par le journal]

Heterogeneity of human haptoglobin α chains detected by two-dimensional gel electrophoresis

“…The appearance of lateral additional spots in 2D gels is often due to variation in the carbohydrate content of a chain but there are no carbohydrate groups on the a chains of haptoglobin (Putnam, 1975). Yang & Przybylska (1973) found that one-dimensional isoelectric focusing separated the native protein of human haptoglobin IS-IS subtype into at least eight distinct components.…”

Crystallization of the haptoglobin–hemoglobin complex

“…Of the new approaches, lowering the temperature (Murakami et al, 1998) and crystallization in microgravity appear promising. Microheterogeneity of the Hp±Hb complex (Yang & Przybylska, 1973) arising from the variation in the sialic acid residue content may be responsible for the observed disorder. Since the crystals of the desialated complex components obtained at the time were too small for X-ray examination, more work on the effect of desialation would be warranted.…”

“…They were strongly birefringent, but the pinacoid end faces were convex, indicating disorder. The crystals of Hp±horse Hb obtained from ammonium sulfate (Yang & Przybylska, 1973) were hexagonal, but too small for X-ray examination. Hwang (1979) obtained much larger hexagonal crystals of Hp±human Hb which diffracted to 7 A Ê and their asymmetric unit contained two molecules of the complex.…”

Thermal and Solvent Perturbation as Probes of Conformational Heterogeneity of Haptoglobin 1‐1

“…However, detailed study of the Hp-Hb interaction has led to the determination of a heterogeneity in the binding affinity of Hp for Hb, suggesting the existence of two forms of Hp molecule : Hp" (form with a lower affinity for Hb) and Hp, (form with a higher affinity for Hb) [ 3 ] . Such a heterogeneity in Hp molecules has also been proposed by Yang and Przybylska and by Jacobsen and Fagerhol [4,5] but has not yet been unambiguously described.…”