Alteration of rheological properties of human erythrocytes by crosslinking of membrane proteins.
The crosslinking of membrane proteins of human erythrocytes by diamide (diazene dicarboxylic acid bis(N,N-dimethylamide) ) was quantified by 4% polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. The relation between the crosslinking of membrane proteins and erythrocyte functions (rheological and oxygen transporting) was quantitatively examined. (i) The crosslinking of membrane protein was induced by diamide, without changing the shape and the contents of intracellular organic phosphates (adenylates and 2,3-diphosphoglycerate). The intensity of spectrin 2 in SDS-polyacrylamide gel electrophoresis decreased proportionally to diamide concentration. The percentage decrease in spectrin 2 (using band 3 as an internal standard) was the most appropriate indicator for crosslinking ("% crosslinking'). (ii) The suspension viscosity of erythrocytes increased in proportion to the percentage of crosslinking, in the range of applied shear rates of 3.76-752 s-1. (iii) Erythrocyte deformability (measured by a high-shear rheoscope) was reduced by the crosslinking. The change was detectable even at 5% crosslinking. (iv) Rouleaux formation (measured by a television image analyzer combined with a low-shear rheoscope) was inhibited by the crosslinking. The inhibition was also sensitively detected at more than 5% crosslinking. (v) Hemoglobin in erythrocytes was chemically modified by higher dose of diamide (probably by the binding of diamide with sulfhydryl groups). Also the oxygen affinity of hemoglobin increased and the heme-heme interaction decreased. (vi) The reduction of the crosslinking of membrane proteins by dithiothreitol apparently reversed the intensity of spectrin bands in SDS-polyacrylamide gel electrophoresis and the erythrocyte functions (the suspension viscosity and the deformability), though not completely.
D001391:Azo Compounds / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D003958:Diamide / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000648:ultrastructure*; D004912:Erythrocytes / Q000187:drug effects / Q000502:physiology; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification; D010108:Oxyhemoglobins / Q000378:metabolism; D014783:Viscosity
D001391:Azo Compounds; D003432:Cross-Linking Reagents; D006454:Hemoglobins; D008565:Membrane Proteins; D010108:Oxyhemoglobins; D003958:Diamide; D004229:Dithiothreitol