Amino acid titration curves--misshapen or mislabeled?

Titration curves are frequently used to illustrate the change in charge of amino acids with pH, and as such are a fundamental part of most biochemistry courses. We have noticed that the graphs of titration curves appearing in many biochemistry textbooks are drawn incorrectly near the origin of the titration. This article demonstrates that this curve is misshapen, or, alternatively, that the axis of the graph is incorrectly labelled.

The influence of salt on the aggregation state of spectrin from bovine erythrocyte membranes.

Sedimentation velocity and gel filtration experiments have been performed with bovine spectrin over a wide range of neutral salt concentrations. Increasing salt concentrations tend to increase both the sedimentation coefficient of spectrin and the elution volume of the protein from 4% agarose columns. No conformation change can be detected by means of optical rotation measurements as the salt concentration is raised. The results are incompatible with the hypothesis that salt causes the aggregation of spectrin, but are consistent with the existence of marked charge effects operative at low salt concentrations. In support of the charge effect hypothesis, acidic groups have been detected on the agarose gels, and ion-exclusion behaviour on the column has been observed with other proteins of similar size.

Physico-chemical characterization of the spectrin tetramer from bovine erythrocyte membranes.

The tetramer of bovine spectrin has been purified and characterized in terms of its hydrodynamic and optical properties. (1) The molecular weight, from both sedimentation equilibrium and sedimentation velocity/diffusion measurements, is close to one million. (2) The hydrodynamic properties suggest a highly expanded but basically symmetrical molecule of Stokes radius 200 A. (3) Optical rotatory dispersion measurements indicate a high degree of order in the tertiary structure of the molecule. These results are not consistent with the assumption that is often made, that the spectrin molecule is a long fibrous rod.

Proteins of the camel erythrocyte membrane.

Electrophoresis on polyacrylamide gels containing dodecyl sulphate has revealed that the major proteins of the camel erythrocyte membrane are similar to those of the human and bovine species in both electrophoretic mobility and relative abundance. The major difference lies in the major intrinsic membrane protein of molecular weight approx. 100 000. In the camel, this protein has a higher apparent molecular weight than in the human and bovine species. The very high molecular weight water-soluble protein "spectrin" appears to be very tightly bound to the camel erythrocyte membrane, and is only partially extracted after prolonged low ionic strength dialysis. Total release of spectrin is only achieved by means of more drastic treatment such as incubation with urea, guanidine hydrochloride, sodium hydroxide of p-chloromercuribenzoate. Concurrent with the total release of spectrin, the camel cells undergo a shape change from flat ellipsoids to spheres, suggesting an important shape-maintaining role for spectrin in the erythrocytes of this species.

Solubilisation of human erythrocyte band 4.1 protein in the non-ionic detergent Tween 20.

Extraction of spectrin-depleted erythrocyte membranes with the non-ionic detergent Tween 20, in a 0.1 M glycine-NaOH buffer (pH 9.8) leads to the solubilization of band 4.1 and the sialoglycoproteins. The comigration of band 4.1 with the sialoglycoproteins in gel filtration and detergent-free electrophoresis indicated that these proteins may be associated as complexes of high molecular weight. Although treatment of intact membranes with Tween 20 under the same conditions does not lead to direct solubilization of proteins, severe disruption of the membranes was observed under phase contrast microscopy. Suspension of the treated membranes in 5 mM phosphate buffer (pH 8.0) leads to the solubilization of band 4.1, spectrin, actin and the sialoglycoproteins. High molecular weight complexes of band 4.1 and the sialoglycoproteins were isolated from these extracts, suggesting a possible interaction between band 4.1 and sialoglycoproteins which may be important for linking the cytoskeleton to the membrane.

A reappraisal of the self-association of human spectrin.

The self-association behaviour of human spectrin has been re-examined through a study of sedimentation equilibrium, sedimentation velocity and gel electrophoresis. In all cases we find evidence for oligomers of spectrin larger than the tetramer, even at low concentration. The data are not consistent with a simple dimer-tetramer model, but instead indicate an open, or indefinite, pattern of association. Although a good fit to the data can be achieved with the isodesmic reaction model, with an equilibrium constant in agreement with the value previously determined for the dimer-tetramer reaction, there is other evidence suggesting that the actual association scheme may be somewhat more complex.

The solubilization of cytoskeletons of human erythrocyte membranes by p-mercuribenzene sulphonate.

The disruption of erythrocyte membrane cytoskeletons brought about by treatment with p-mercuribenzene sulphonate (PMBS) has been followed by measurements of turbidity and the binding of 203Hg-labelled PMBS. After pretreatment with N-ethylmaleimide to block readily reactive sulphydryl groups, incubation with [203Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal pKa value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups.

Solubilization and denaturation of monomeric actin from erythrocyte membranes by p-mercuribenzenesulfonate.

Solutions of p-mercuribenzenesulfonate extract the peripheral proteins from the red cell membrane in a water-soluble form. Low concentrations of the reagent selectively solubilize actin, while at higher concentrations, spectrin, ankyrin and bands 4.2 and 4.1 are extracted. After brief exposure to the reagent, followed by displacement of the mercurial with dithiothreitol or 2-mercaptoethanol, the soluble actin is capable of inhibiting DNAse I activity. With prolonged exposure or with higher concentrations of the reagent, the ability to inhibit DNAse is gradually lost. The kinetics of both the release of actin capable of DNAse inhibition and the subsequent loss of that capability are pseudo-first-order with respect to time, but show second-order dependence on the concentration of mercurial. These data suggest that dissociation of the actin from protofilaments in the cytoskeleton requires exposure of more than one sulfhydryl group to the reagent. Subsequent inactivation also appears to be dependent on the reaction of further multiple sulfhydryl groups, possibly in buried regions of the actin molecule.

The 'hollow cylinder' protein of erythrocyte membranes.

The 'hollow cylinder' protein (Harris, J.R. (1968) Biochim. Biophys. Acta 150, 534-537) has been purified from human erythrocyte membranes. The molecular weight of the native protein, as determined by analytical ultracentrifugation, was found to be 747,000. By means of sodium dodecyl sulphate gel electrophoresis, the purified protein was shown to be composed of three different low molecular weight polypeptides of average molecular weight 25,000. This study provides convincing evidence that the spectrin tetramer is not responsible for the characteristic electron microscopic appearance of the hollow cylinder protein.

Salt and temperature-dependent conformation changes in spectrin from human erythrocyte membranes.

ORD and CD measurements of spectrin, in both the dimer and tetramer association state, indicate a high proportion of alpha-helix in this protein. At temperatures below 27 degrees C and in 0.1 M NaCl, the tetramer has an apparent helix content of 73% and the dimer, 68%. The conformation of both states is dependent on salt concentration and temperature. Low ionic strength solutions of spectrin display lowered sedimentation coefficients and a decreased apparent helix content, indicating perhaps a slight refolding and expansion of the molecule. In addition, spectrin in low ionic strength solutions undergoes a broad temperature-dependent transition spread from 20 to 50 degrees C, while in the presence of salt the transition is sharp and centered on 49 degrees C. The temperature-dependent changes in low ionic strength solutions appear to parallel the dissociation of tetramer to dimer.

The effects of ionic strength on the self-association of human spectrin.

The self-association of human spectrin has been studied by means of sedimentation equilibrium in the analytical ultracentrifuge at pH 7.5 and over a range of ionic strength from 0.009 to 1.0 M. Increasing ionic strength above 0.1 M reduces the equilibrium constants for all of the measurable steps in the self-association reaction. These results support the concept of charge-charge interactions stabilizing the tetramer and higher oligomers with respect to the heterodimer. In addition, increasing ionic strength brought about a dissociation of the heterodimer to component polypeptide chains. Dissociation to the heterodimers is also enhanced with a decrease in ionic strength below 0.05 M. This low ionic strength-dependent dissociation is consistent with generalised electrostatic repulsion; however, this effect also correlates with some loss of alpha-helical content as revealed by circular dichroism. The secondary, tertiary and quaternary structures may all be partially disrupted by electrostatic free energy at low ionic strength.

The incorporation of 32 P into spectrin aggregates following incubation of erythrocytes in 32 P-labelled inorganic phosphate.

32P was incorporated into spectrin by incubation of fresh erythrocytes with 32Pi and glucose. The dimer and tetramer aggregates revealed only covalently-bound incorporation of phosphorus, while a higher aggregate of spectrin revealed both covalent and non-covalent incorporation. The specific activity of the covalently-bound phosphorus in all oligomers was identical, suggesting that the state of association is independent of phosphorylation. The non-covalent incorporation was shown to be due to the association of ATP with this higher aggregate. The nucleotide appers not to be bound directly to spectrin but rather to component 5 (erythrocyte actin) which is also found to be associated with this highly aggregated spectrin structure.

Quantitative analysis of the amino-terminal residues of spectrin by use of the transamination reaction.

The metal ion-catalysed transamination reaction has been examined as a means of quantitative amino-terminal analysis of proteins. Application of this method to the erythrocyte membrane protein, spectrin, showed that this protein contained a single amino-terminal residue per 240,000 daltons. This value supports the hypothesis that spectrin is comprised of two polypeptide chains of approx. 220,000 and 250,000 daltons, respectively.

Binding of an 80 000 dalton trypsin fragment of spectrin to intact spectrin.

A specific and saturable interaction of an 80 000 dalton tryptic fragment of spectrin with intact spectrin has been detected. When spectrin was incubated with 125I-labelled 80 kDa fragment at 37 degrees C in the presence of 0.1 M NaCl, acrylamide gradient gel electrophoresis showed the presence of bands in addition to the usual spectrin dimer and tetramer and the 80 kDa fragment. These bands correspond to 5.6 X 10(5) daltons (dimer + fragment), 1.04 X 10(6) daltons (tetramer + fragment) and 1.52 X 10(6) daltons (hexamer + fragment). Measurement of radioactivity showed that these additional bands contained the labelled fragment. Maximal binding capacity of the 80 kDa fragment was approximately 170 micrograms fragment per mg spectrin, corresponding to 1 mol fragment per mol spectrin dimer.

The action of organic mercurials on the erythrocyte membrane.

The solubilisation of proteins from erythrocyte membranes by treatment with organic mercurials has been studied with different species. The marked solubilisation previously reported for human membranes does not seem to be a general phenomenon. All of the other species examined showed less than 50% of the solubilisation shown by human membranes. The protein-solubilising effect seems to be dependent on hydrophobic mercury derivatives carrying a net negative charge. Uncharged compounds like phenylmercuric acetate blocked the effect, although N-ethylmaleimide and iodoacetamide did not. With the aid of radioactively labelled compounds, and of atomic absorption spectrophotometry, the proteins reactive towards the mercurials were identified. The major integral protein, band 3, was the major protein capable of binding the mercurial. Reaction with the mercurial appears to disrupt interaction of band 3 with bands 2.1 and 4.2, allowing dissociation of the cytoskeleton from the membrane. In addition, band 4.9 was also found to react with the mercurials, possibly resulting in disruption of the cytoskeleton.

Hydrodynamic characterization of the heterodimer of spectrin.

The hydrodynamic properties of the spectrin dimer have been examined. The S20,w value of 9.3 S and the D20,w value of 1.75 x 10(-7) cm2 x s-1 yield a molecular weight of 470 000, in good agreement with the value from sedimentation equilibrium of 460 000. The frictional ratio of 2.3 and the intrinsic viscosity of 36 ml/g are not consistent with a compact, globular structure, but the value of the parameter Ks/[eta], 1.15, is not consistent with a rigid rod model. The most appropriate model, consistent also with data from other laboratories, is of a flexible, kinked rod-like molecule.

Purification of a trypsin-insensitive fragment of spectrin from human erythrocyte membranes.

When spectrin is treated with trypsin, a series of polypeptide fragments is generated, One particular fragment having an approximate molecular weight of 80 000 constitutes 18% of the trypsin-digested mixture and is trypsin-insensitive. This fragment has been isolated and purified by gel filtration followed by ion-exchange chromatography. The molecular weight of the fragment, as seen from sedimentation equilibrium measurements and from gel electrophoresis, both in the presence and absence of detergent, is close to 80 000. There was no evidence of self-association under the conditions used. Changes in the specific rotation at 365 nm were used to detect temperature-dependent conformation changes in the fragment and to compare these changes with those in the intact spectrin molecule. The fragment undergoes temperature-dependent transitions centered at 46 and 58 degrees C, similar to those in intact spectrin (49 and 55 degrees C). Although the thermal transitions exhibited by intact spectrin are markedly salt-dependent, those shown by the fragment are not. ORD (optical rotary dispersion) measurements indicate 53% apparent alpha-helix in the fragment, compared to 68% in intact spectrin. Antibodies raised against the fragment cross-react only with band 1, the largest polypeptide of spectrin, indicating that the fragment is derived from band 1.

The dissociation of peripheral proteins from erythrocyte membranes brought about by p-mercuribenzenesulfonate.

The organic mercurial p-mercuribenzenesulfonate in 5 mM phosphate buffer (pH 8.0) solubilized ankyrin, bands 4.1 and 4.2, and glyceraldehyde-3-phosphate dehydrogenase from spectrin-depleted erythrocyte membranes. Glyceraldehyde-3-phosphate dehydrogenase was the protein most readily solubilized, being almost completely extracted by 0.5 mM reagent. The solubilization of ankyrin was similar to that of band 4.2, both showing maximal solubilization with 1.0 mM reagent. Band 4.1 was not appreciably solubilized below 2.5 mM p-mercuribenzenesulfonate. N-Ethylmaleimide did not itself solubilize proteins from ghosts or spectrin-depleted vesicles, and pretreatment at low temperature by 4 mM N-ethylmaleimide did not prevent subsequent solubilization by the mercurial. However, pretreatment at 37 degrees C with N-ethylmaleimide inhibited subsequent solubilization of ankyrin and band 4.2 by the mercurial and also resulted in the loss of binding of 1 mol mercurial per mol band 3. These data suggest that release of ankyrin and band 4.2 from the membrane by mercurial is linked to modification of band 3 by the reagent. After incubation of intact erythrocyte membranes with 0.1 M NaCl, treatment with p-mercuribenzenesulfonate selectively solubilized actin from the membranes. The resulting actin-depleted membranes did not vesiculate, but became spherical and lost their biconcave shape. Fragmentation was observed after subsequent removal of spectrin at low ionic strength.

A water-extractable Ca2+-atpase from erythrocyte membranes.

The Ca2+- and Mg2+-stimulated ATPase activities present in low ionic strength extracts of erythrocyte membranes have been separated from each other. The Ca2+-ATPase appears to be associated with particulate meterial which could be sedimented by high-speed centrifugation. The pellet obtained was composed mainly of components 1, 2, 4.5, 5 and 7. A soluble protein from the band 3 region, known to be responsible for the Mg2+-ATPase activity, was not detected in this pellet.

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes.

A water-soluble Mg2+-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567-576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The Km value for ATP was 1 mM and apparent Km for Mg2+ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd2+ and Zn2+ salts, p-chloromercuribenzoate and N-ethylmaleimide, known inhibitors of membrane endocytosis.