The ANS- (1-anilino-8-naphthalene sulfonate) anion is strongly, dominantly bound to cationic groups of water-soluble proteins and polyamino acids through ion pair formation. This mode of ANS- binding, broad and pH dependent, is expressed by the quite rigorous stoichiometry of ANS- bound with respect to the available summed number of H+ titrated lysine, histidine, and arginine groups. By titration calorimetry, the integral or overall enthalpies of ANS- binding to four proteins, bovine serum albumin, lysozyme, papain, and protease omega, were arithmetic sums of individual ANS(-)-polyamino acid sidechain binding enthalpies (polyhistidine, polyarginine, polylysine), weighted by numbers of such cationic groups of each protein (additivity of binding enthalpies). ANS- binding energetics to both classes of macromolecules, cationic proteins and synthetic cationic polyamino acids, is reinforced by the organic moiety (anilinonaphthalene) of ANS-. In a much narrower range of binding, where ANS- is sometimes assumed to act as a hydrophobic probe, ANS- may become fluorescent. However, the broad overall range is sharply dependent on electrostatic, ion pair formation, where the organic sulfonate group is the major determinant of binding.
The cytoskeleton underlying the membrane of erythrocytes is thought to control changes in cell shape such as the diskocyte to the echinocyte. Since the binding of lectins to the transmembrane protein glycophorin blocks the cell shape change, we have proposed that the cytoplasmic end of glycophorin is linked to the cytoskeleton. Here we show that the cytoskeletal protein known as band 4.1 specifically associates with the cytoplasmic domain of glycophorin on inside-out erythrocyte membrane vesicles and also with glycophorin reconstituted into phosphatidylcholine vesicles. The binding of band 4.1 to glycophorin is saturable and inhibitable by antibodies which bind specifically to the cytoplasmic domain of glycophorin. We therefore believe that band 4.1 protein provides the link between glycophorin and the cytoskeleton.
When an azo dye is converted from the trans to the cis form, there occur two kinds of changes: (1) a change in geometry, with accompanying absorption spectral changes (photochromism), and (2) a change in intermolecular interactions and crystal packing in which the molecule can engage. For example, cis azobenzene derivatives frequently melt at a considerably different temperature from that of the corresponding trans isomers.' If such chromophores were parts of polymers, or bound to them, it seemed possible that light energy might be made to influence the conformation of the polymers. This seems reasonable from a thermodynamic standpoint. Non-cross-linked polyelectrolytes, for example, if capable of undergoing enthalpy-dominated conformation changes, usually do so upon acquiring 10-100 kcal of electrostatic energy per mole for an ordinary sized (mol wt 104 to 106) macromolecule.2 Near-ultraviolet and visible light possess 40-90 kcal of energy per einstein. Upon absorption of it, a considerable fraction may be used in activation processes, but in the case of azobenzene itself, there is a residue of potential energy difference stored in the isomers which amounts to 9.9 kcal3 (the trans form being the most
The lectin wheat germ agglutinin (WGA) is an unusually effective agent in controlling both the forward and reverse reactions of the reversible morphology conversion discocyte~:± echinocyte for the human erythrocyte . Under conditions severe enough to drive the reactions to completion in either direction without the lectin, WGA is able to stabilize both these morphologies and to fully prevent conversion of either morphology . The lectin can quantitatively block both reactions . The ability of WGA to carry out these functions has no obvious rate limitation . Its effectiveness depends mainly on its binding stoichiometry, particularly toward the transmembrane glycoprotein, glycophorin . The critical binding stoichiometries for both the lectin and the echinocytic agent were determined in relation to the binding isotherms using "'I-labeled WGA and 35 S-labeled dodecyl sulfate . There appear to be two principal stoichiometries for WGA binding that are important in its control of erythrocyte morphology . The first stoichiometry marks the threshold of obvious protection of the discocyte against strong echinocytic agents such as detergents and, likely, is simply a 1 :1 stoichiometry of WGA : glycophorin, assuming currently recognized values of 3-5 X 101 ' copies of glycophorin per cell. The second important stoichiometry, whereby the cell's morphology is protected against extremely severe stress, involves binding of -4-5 WGA molecules per glycophorin . The controls that WGA exerts can be instantly abolished by added N-acetylglucosamine . However, N-acetylglucosamine ligands on the erythrocyte are ofless importance than membrane neuraminic acid residues in enabling WGA to control the cell's morphology, as is shown by comparing intact cells with completely desialated cells . WGA can also be used to produce elliptocytes in vitro, but it does this at levels approaching monolayer coverage of the cell with WGA .Lectins have long been recognized as proteins that agglutinate human erythrocytes . Their agglutination properties have been important tools in detecting several of the blood groups (17) . Less obvious, however, is the extent of the control lectins exert over the discrete morphologies that may be J.
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