The indefinite self-association of lysozyme: consideration of composition-dependent activity coefficients

An improved iterative method for computing association constants from sedimentation equilibrium results obtained with self-interacting protein systems is presented which accounts for the composition-dependence of the activity coefficients of all oligomeric species. The method is based on the calculation of virial coefficients from covolume and charge considerations, the statistical mechanical basis of which is discussed in relation to the DLVO theory. The method is applied to results obtained with lysozyme in diethylbarbiturate buffer of pH 8.0 and ionic strength 0.15 at 15°C. It is shown that these results, encompassing a range of total solute concentration up to 19.7 g/liter are consistent with self-association patterns comprising either a monomer-dimer-trimer system or an isodesmic indefinite self-association of the monomer, the latter being favored. A firmer distinction between these possibilities is sought on the basis of the dependence of the weight-average partition coefficient, determined by frontal gel chromatography, on total solute concentration (up to 56.6 g/liter). This analysis accounts for the composition-dependence of the ratio of the activity coefficients of partitioning monomer in mobile and stationary phases. It is concluded that all results are consistent with an indefinite self-association of lysozyme governed by a single association constant of 4.61 × 10² liter/mole.     

Sedimentation coefficients of self-associating species: I. Basic theory

Under the same solution conditions, the apparent weight average sedimentation coefficient, s_wa, and some quantities obtained from it can be combined with the equilibrium constant or constants, K_i, and the monomer concentration, c_I, obtained from sedimentation equilibrium, light scattering or osmotic pressure experiments on the same self-associating solute, so that the individual sedimentation coefficients, s_i, of the self-associating species, and also the hydrodynamic concentration dependence parameter,g or $\text{g}$, can be evaluated. Using two different models for the hydrodynamic concentration parameter, four different methods are presented for the evaluation of the s_i's. Methods for evaluating g or $\text{g}$, once the s_i's are known, are presented. A method for obtaining the number average sedimentation coefficient, s_N, and its application to self-associations is presented. Methods are shown for the evaluation of Z average properties, x_zc, as well as number average properties,x_Nc, of a self-associating solute from its weight average properties, x_wc.     

Polymerization studies on protein from the dahlemense strain of tobacco mosaic virus: Light scattering and related studies

Light-scattering and related studies on protein of Dahlmense strain of tobacco mosaic virus (DTMV) show that its polymerization characteristics are considerably different from those of TMV protein. At pH 6.0 in phosphate buffer (I = 0.1), the extent of polymerization of DTMV protein is greater than that of TMV protein, they are nearly the same at pH 6.25, and that of DTMV protein is less than that of TMV protein at pH 6.5. At pH 7.0 and 7.5, DTMV protein polymerizes more readily than TMV protein. Similar studies in phosphate buffer (I = 0.05) show that the extent of polymerization for DTMV protein is less than that of TMV protein at pH 6.0 and almost negligible at pH 6.25. Acid-base titration studies show that, upon temperature-mediated polymerization, about 2 H⁺ ions are bound per monomer of DTMV protein at pH 6.O.Electron microscope studies show that DTMV protein exists at room temperature as double discs and polymerized rods in phosphate buffer at pH 7.5, I = 0.1; at pH values below 6.5, DTMV protein is entirely in the form of polymerized rods. Velocity sedimentation studies of DTMV protein at room temperature are in agreement with these findings. At low temperatures, except at pH 7.5, most of the material sedimented with an s value of around 25 S. Thus, at low temperatures, except at pH 7.5, DTMV protein in solution is in the form of particles the size of double discs with an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 596,000 g/mole or even larger. Therefore, temperature-mediated polymerization of DTMV protein at pH values below 6.5 in phosphate buffer (I = 0.1) and below 6.25 in phosphate buffer (I = 0.05) involves particles at least as large as double discs as the starting material.     

Exclusion chromatography of concentrated hemoglobin solutions: Comparison of the self-association behavior of the oxy and deoxy forms of the α2β2 species

Theory pertaining to the interpretation of partition chromatography results obtained with self-associating protein systems studied at high total concentrations is extended to permit consideration of situations in which both monomeric and dimeric states partition. This development, which includes considerations of thermodynamic nonideality effects, permits a quantitative correlation of human oxyhemoglobin results reported previously and obtained in this work employing a different stationary matrix of controlled-pore glass beads. The two sets of results, obtained at pH 7.3 and 20°C- indicate that the α₂β₂ species of oxyhemoglobin self-associates. Two types of association pattern, discrete dimerization and an indefinite self-association, are examined. This is done for a realistic range of values for the radius, r, of the effective hard sphere appropriate to the calculation of the covolume of the α₂β₂ species in the assessment of the thermodynamic nonideality contribution. Assessed values of the isodesmic association constant range from 66 = 23 M ^?1(r = 2.84 nm) to 154 = 26 M^?1' (r = 3.13 nm). This mode of indefinite association is marginally favored over a dimerization when the larger value of r is considered, the two patterns becoming virtually indistinguishable for the lower value of r. Partition chromatography results are also presented for human deoxyhemoglobin up to a total concentration of 225 $\text{g}\text{I}$, and are analyzed in a similar fashion to show that the indefinite self-association pattern is favored, governed by an isodesmic constant in the range 91 = 9 M^?1(r = 2.84 nm) to 223 = 84 M^?1 (r = 3.13 nm). Comparison of the constants assessed for the oxy and deoxy systems permits discussion of the concept that oxygen binds preferentially to the α₂β₂ species of deoxyhemoglobin in comparison with its polymers.     

Analysis of chromatin repeat units in logarithmically and stationary growing cells of Paramecium aurelia and Tetrahymena pyriformis.

We have used micrococcal nuclease as a probe of the repeating structure of chromatin isolated from the macronuclei of logarithmically and stationary grown $\text{Paramecium aurelia}$ and $\text{Tetrahymena pyriformis}$. For both these lower eukaryotes, the monomer size is shown to vary depending on the stage in the growth cycle. $\text{P. aurelia}$ exhibits a monomer size of 153±7 bp and 178±6 bp and $\text{T. pyriformis}$ 207±10 bp and 230±10 bp in logarithmic and stationary cells, respectively. Both exhibit a nucleosome size of 140 bp. We discuss the possible association of these changes with histone content and nuclear activity changes, and also a possible reason for the divergence from the size pattern of monomer repeats seen in lower eukaryotes by $\text{T. pyriformis}$.     

Graphical analysis of nonideal monomer N-mer, isodesmic, and type II indefinite self-associating systems by equilibrium ultracentrifugation.

A graphical procedure is described by which one can obtain in principle the monomer molecular weight, stoichiometry, equilibrium constant, and second virial coefficient of nonideal monomer N-mer, isodesmic, and type II indefinite self-associating systems. In addition, a method is presented for obtaining both the equilibrium constant and the second virial coefficient from the maximum in a plot of apparent molecular weight vs. concentration if the monomer molecular weight and stoichiometry are known. The usefulness and limitations of the methods are discussed, as well as the quality and range of data required for determination of the relevant parameters. The techniques described are applicable to analysis of self-associating systems by osmotic pressure and light scattering, as well as equilibrium ultracentrifugation measurements.     

Purification of estradiol-17 beta dehydrogenase from human placenta by affinity chromatography

Estriol 16-hemisuccinate has been synthesized and covalently attached to Sepharose through 1,5-diaminopentane. A crude preparation of estradiol-17β dehydrogenase from human placenta was adsorbed on the gel. After extensive washing, the enzyme was eluted by $\text{M}$ hydroxylamine in 0.1 $\text{M}$ potassium phosphate buffer (20–50% glycerol), pH 7, at room temperature. An apparently homogeneous enzyme with a specific activity of 7.2 U/mg (82% recovery) was obtained. It is stable for weeks in the eluting buffer. The hydroxylamine can be removed by passing the enzyme solution over a Sephadex G-100 column or by dialyzing it against 0.1 $\text{M}$ potassium phosphate buffer containing 20% glycerol. This one-step process makes purification of the enzyme simple and easy.     

Some association properties of bovine SH-k-casein

(1) It is shown ibal ^k-casein association is characterized hv a critical micelle concentration which decreases as the ionic strength is increased. (2) The ^k-casein polymer molecular weight was calculated from the weight-average apparent molecular weight by taking into consideration the monomer concentration and the excluded volume. The degree of polymerization is 30 and does not depend on ionic strength between 0.1 and 1 M. (3) The non-electrical contribution to the standard free energy of association is ?38 $\text{kJ}\text{mol}$ monomer. The electrical part is small: 1–2 $\text{kJ}\text{mol}$ monomer depending on the ionic strength and ^k-casein genetic variant. (4) The limitation of size and the size itself of the ^k-casein polymer can be explained by the theory of self-assembly of virus particles by Caspar and Klug (D.L.D. Caspar and A Klug. Cold Spring Harb. Symp. Quant. Biol. 27 (1962)1). (5) Extending this theory to casein micelle assembly, it is predicted that micelles are distributed preferentially over a restricted number of sizes.     

Calorimetric determination of polymerization enthalpy for bacterial flagellin (Salmonella)

The polymerization of bacterial flagellin protein (Salmonella strain SJ814) into flagellar filaments has been found by direct calorimetric measurement to be $\text{exothermic}$ at 25° in .15M KCl, pH 6.8 with a ΔH of ?12.7 ± 0.6 kcal per mole of monomer polymerized. The calorimetric result at 25° contrasts sharply with the endothermic ΔH of +38 kcal/mole inferred from temperature dependence of the critical monomer concentration near 40°C. Comparison between these two values implies that unless a different mechanism of polymerization prevails at the two temperatures the heat capacity change for flagellin polymerization may be as large as 3.3 kcal/mole deg.     

Alcohol-induced potentiation of the neurally evoked contractions of the retractor unguis muscle of the locust, Schistocerca gregaria

Each of a series of seven monohydroxyl alcohols caused an increase in the force of the neurally evoked contractions of the innervated retractor unguis muscle isolated from the metathoracic femurs of the locust, Schistocerca gregaria. The negative logarithm of equipotent concentrations of the alcohols was proportional to the logarithm of the partition coefficient (1-octanol/water) of the alcohols: $\text{log}\text{1}\text{C}\text{= 1·282}\text{log}\text{P+1·684}$, where C is concentration, and P is the partition coefficient.Ethylene glycol and glycerol did not potentiate the contractions, possibly due to their very low lipophilic character. Acetone (10^?2 M) and benzene (10^?5 M) also potentiated the neurally evoked contractions of the insect muscle.     

The effect of sodium dodecyl sulfate on the structure and function of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) has been shown to exist as a dimer of molecular weight of 23,000 in 25 mm phosphate buffer, at pH 7.0 (the ionic strength 0.1 m with NaCl), 25.0 °C in the concentration range of 0.01–10 mg/ml. In the present paper, the effects of an anionic detergent, sodium dodecyl sulfate (SDS), on the structure and function of SSI has been examined, [a]The molecular weight of SSI was measured in the SDS solution with the sedimentation equilibrium method of the multicomponent-polydisperse system under the conditions described above, and thereby it has been shown that SSI dissociates into monomers with SDS of 0.03–0.12% ($\text{w}\text{v}$) when the concentration of SSI is 1.00 mg/ml (87.0 μm as monomer), [b]As SSI dissociates into monomers, there were observed blue-shift troughs at 293 nm and 300 nm due to a tryptophyl residue and a red-shift of phenylalanyl residues in the absorption difference spectrum induced by the binding of SSI and SDS. [c] The inhibitory activity of SSI against subtilisin BPN′-catalyzed hydrolysis of p-nitrophenyl acetate was measured under the conditions that SSI is in monomer in the SDS solution. Unexpectedly half of the inhibitory activity of SSI against subtilisin BPN′ is lost in the SDS solution.     

Interactions of lens proteins. Self-association and mixed-association studies of bovine alpha-crystallin and gamma-crystallin

Concentrated solutions of calf alpha-crystallin (up to 45 g/l) and gamma-crystallin (up to 67 g/l) were subjected to frontal exclusion chromatography at pH 7.3, ionic strength 0.17 and 20 degrees C. The experimental concentration dependence of the weight-average partition coefficient was compared with theoretical expressions, which include considerations of thermodynamic non-ideality effects, for the concentration dependence of a single solute and of a solute undergoing reversible self-association. Two types of association pattern were examined, discrete dimerization and indefinite self-association. The partition chromatography results are consistent with an indefinite self-association of gamma-crystallin, governed by an isodesmic association constant of 6.7 X 10(-3) l/g. alpha-Crystallin appears to self-associate either very weakly, with a maximal association constant of 0.9 X 10(-3) l/g, or not at all; the distinction depends on the assessment of the non-ideality coefficients. The consequences of excluded volume effects on these self-association equilibria at high total protein concentration are discussed. Mixtures of alpha-crystallin and gamma-crystallin were analyzed by frontal exclusion chromatography (up to 14 g/l) and sedimentation velocity (up to 115 g/l): no interaction was observed.     

The effect of anaesthetic-like molecules on the phase transition in smectic mesophases of dipalmitoyllecithin I. The normal alcohol up to C = 9 and three inhalation anaesthetics

The effect of the normal alcohols (up to $\text{C = 9}$) and three clinically used anaesthetics, on the crystalline-liquid crystalline phase transition in $\text{1,2-}\text{dihexadecyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphorylcholine}$ have been studied. A one-degree depression was produced by a 4.4% concentration in the membrane of $\text{n-}\text{octanol}$ and $\text{n-}\text{nonanol}$ agreeing well with the value calculated from the temperature and enthalpy of the transition. It is also shown that the relationship between the partition coefficient $\text{P}$ and the water solubility $\text{S (P · S = 2)}$, holds for the solutes investigated here. The experimental method described offers a simple way of assessing the anaesthetic potency of a wide range of compounds.     

Measurement of osmotic second virial coefficients by zonal size-exclusion chromatography

Numerical simulation of protein migration reflecting linear concentration dependence of the partition isotherm has been used to invalidate a published procedure for measuring osmotic second virial coefficients (B₂₂) by zonal exclusion chromatography. Failure of the zonal procedure to emulate its frontal chromatographic counterpart reflects ambiguity about the solute concentration that should be used to replace the applied concentration in the rigorous quantitative expression for frontal migration; the recommended use of the peak concentration in the eluted zone is incorrect on theoretical grounds. Furthermore, the claim for its validation on empirical grounds has been traced to the use of inappropriate B₂₂ magnitudes as the standards against which the experimentally derived values were being tested.     

Kinetics of hydrolysis to tetraols and binding of benzo(a)pyrene-7,8-dihydrodiol-9,10-oxide and its tetraol derivatives to DNA. Conformation of adducts

When the major reactive metabolite of benzo(a)pyrene, $\text{trans}$ -7,8-dihydroxy - $\text{anti}$-9,10-epoxy -7,8,9,10-tetrahydrobenzo(a)pyrene ($\text{anti}$-BPDE) is incubated with DNA in aqueous solution at 25°C, both covalent binding and hydrolysis of $\text{anti}$-BPDE to its tetraols occur. Using fluorescence and absorption spectroscopy it is shown that hydrolysis of $\text{anti}$-BPDE is markedly accelerated by DNA. In the presence of 5A₂₆₀ units of DNA per ml in cacodylate buffer solution, at an initial concentration of DNA phosphate/$\text{anti}$-BPDE ratio of 100, both the extent of covalent binding to DNA ( < 7% of the total $\text{anti}$-BPDE initially present) and hydrolysis of $\text{anti}$-BPDE reach their maximum levels within less than five minutes after mixing. Absorption and electric linear dichroism spectra indicate that the tetraols bind non-covalently to DNA by an intercalation mechanism, whereas the covalent product displays the characteristics of an externally bound complex.     

Evidence for a quaternary structure change in the cooperative binding of cyanide to ferrihemoglobin A.

At pH 6.5 in a 0.05 $\text{M}$ bis-Tris-0.1 $\text{M}$ Cl^? buffer, tetra aquo ferrihemoglobin A (HbA⁺) binds CN^? with a Hill coefficient of n = 1.4. The Hill coefficient increases slightly and the average CN^? affinity decreases in the presence of excess spin labeled triphosphate (SLTP). This is probably the result of the finding that the SLTP exhibits a twofold higher affinity for HbA⁺ than for tetra cyano HbA⁺. Over the course of heme saturation with CN^?, a certain fraction of the SLTP is specifically released. This shows linkage between organic phosphate binding and heme ligation. These findings bear a marked resemblance to the ligand binding phenomena in hemoglobin A (HbA) and provide good evidence that under these experimental conditions, HbA⁺ is undergoing a quaternary conformation change as the hemes become saturated.     

Rapid down regulation of insulin receptors in adipocytes artifact of the incubation buffer

Rat adipocytes were incubated with 15 nM insulin in different buffers at 37°C. The cells were washed and reincubated at 16°C in the presence of 18 pM A14-[¹²⁵I]monoiodoinsulin to determine the insulin receptor concentration. After incubation for 2 h in Tris buffer the binding decreased to about 30 %, whereas no decrease was found after incubation in Hepes, phosphate or bicarbonate buffers. Binding of tracer insulin reached a constant level by 45 min in Hepes buffer at 37°C, whereas it continued to increase in Tris buffer. Washout of tracer insulin after incubation in Tris buffer at 37°C showed a large, slowly dissociable fraction. It is suggested that the rapid down regulation of insulin receptors $\text{in}\text{vitro}$ is an artifact of the Tris buffer and that the phenomenon is due to a slowly reversible occupancy of a receptor pool with unlabelled insulin.     

Determination of zinc by flameless atomic absorption spectrophotometry

The flameless atomic absorption method described here is a simple, rapid, accurate microtechnique for determining zinc in aqueous solutions, serum, or urine. It requires no sample pretreatment, only 1.0 μl of sample per determination, no correction for viscosity differences between sample and standard solutions, and is not subject to ionic or organic interference. The average recovery of added zinc in serum is 97.5% and in urine is 97.6%. The values obtained for serum (mean ± SD: 94.6 ± 11.0 μAg/100 ml; N = 25) and urine (range: $\text{sol}\text{600–1000 μg}\text{24}\text{hr}$; N = 4) are comparable to the values reported in the literature. The coefficient of variation was less than 5.0% in all cases. The qualitative concentration limit was $\text{0.009}\text{μg}\text{100}\text{ml}$. The techniques and instrumentation described are also applicable to a number of trace minerals of common interest.