Phil G. Squire

Hydrodynamics and protein hydration.

We have critically evaluated hydrodynamic data from 21 proteins whose molecular dimensions are known from X-ray crystallography. We present two useful equations relating the molecular weights and sedimentation coefficients of globular proteins. The hydrodynamic data combined with data for small molecules from the literature indicate that failure of the Stokes equation occurs only for molecular weights <850. Calculated hydration values for the 21 proteins have a mean value and standard deviation of 0.53 ± 0.26 g H2O/g protein. Furthermore, statistical arguments indicate that only 5.3% of the variance is due to experimental error. The mean value and especially the dispersion of values are in sharp contrast to the values 0.36 ± 0.04 obtained by others from nmr measurements on frozen protein solutions. Hydration values calculated from nmr measurements are closely correlated with the number of charged and polar amino acid residues. In contrast to this result, our analysis of the amino acid compositions of the four proteins with the lowest hydration and the four monomeric proteins with the highest shows that the range of values we observe cannot be accounted for on the basis of amino acid composition. In fact there appears to be a weak correlation between the number of apolar residues and hydrodynamic hydration. We therefore conclude that the dispersion must result from variations in fine details of the surface structures of individual proteins. We propose a model of hemispherical clathrate cages which if correct, would account for the differences in the data obtained by these two methods.

Calculation of hydrodynamic parameters of random coil polymers from size exclusion chromatography and comparison with parameters by conventional methods

Abstract A new method of treating data from size exclusion chromatography, originally evaluated for native proteins, has been extended to random coil polymers using data from dextran and polyethylene glycol as specific examples. Columns are calibrated with globular proteins of known molecular weight. Multiplication by a constant transforms the abscissa to molecular radii. The constants in the equation r = gM Z are then evaluated directly from the size exclusion chromatographic data and compared with the corresponding constants obtained from viscometry, sedimentation velocity and light scattering, relating Stokes radius, root mean square end-to-end distance and radius of gyration to molecular weight.

High-performance size exclusion chromatography of sea worm chlorocruorin and other large proteins, viruses and polysaccharides on a TSK G5000 PW preparative column

Abstract The elution parameters of large enzymes, viruses, ribosomes and other “supramolecular” structures are studied using the preparative TSK G5000 PW type column. The pigmented protein, chlorocruorin, isolated from the sea worm Potamilla leptochaeta , was found to serve as an excellent high-molecular-weight marker for size exclusion liquid chromatography. This is due to its high degree of molecular stability and a molecular weight, found to be 2.9·10 6 by sedimentation velocity analysis, which is located in a zone formed between viruses and enzymes that is largely devoid of macromolecular markers. Calibration constants for this chromatography column are found for both molecular weight and molecular radii. The data found for hydrodynamic molecular radii are further extended to non-globular, swollen macromolecules, such as polysaccharides, using dextran fractions sized by alcohol precipitation.

Size exclusion at the external surfaces of spherical packing used in high-performance liquid chromatography

Experimental observations from several laboratories, including our own, have demonstrated that the elution volume of very large macromolecules or particles shows a small dependence on size even when the packing gels are impermeable to these substances. We call this process “external size exclusion”, but it has also been termed “hydrodynamic chromatography/rd. We have derived a quantitative expression for the phenomenon based on an equilibrium process in which spherical macromolecules of radius r pass through a column filled with uniform spheres of radius R in hexagonal close packing. The final expression gives the ratio of the elution volume Ve of a sphere of radius r to the total volume of the column Vt. The range of practical usefulness is 0.01 < r/R < 0.1. “Premature” elution of aggregated tobacco mosaic virus is explained by the use of this equation.

BOVINE ADRENAL GLUCOSE-6-PHOSPHATE DEHYDROGENASE.

We have developed a new procedure for the purification of glucose-6-phosphate dehydrogenase from bovine adrenals. Three kg of whole adrenals are homogenized and fractionated by precipitation with ammonium sulfate, adsorption and elution from calcium phosphate gel, chromatography on cellulose phosphate, chromatography on DEAE-Sephadex, gel filtration on Sephadex C-J50, and crystallization from ammonium sulfate solution. The procedure requires about three weeks and the yield is 24 mg of enzyme. Approximately half of the original enzyme activity is lost by inactivation in spite of considerable effort to minimize these losses. Analysis of the final product by sedimentation velocity displays a single Gaussian peak, and the boundary spreading can be attributed almost entirely to diffusion, thus a high degree of mass homogeneity is demonstrated. The sedimentation coefficient converted to standard conditions, Szo.w is 9.47S. This is consistent with a molecular weight of 190,000. The enzyme also displays a single band when submitted to electrophoresis on cellulose acetate strips. The activity of 6-phosphogluconate dehydrogenase disappears early in the fractionation procedure.

Bovine adrenal glucose-6-phosphate dehydrogenase III. Control of the activity by ascorbate, substrates, and hydrophobic molecules

Abstract 1. 1. Earlier studies with bovine adrenal glucose-6-phosphate dehydrogenase demonstrated that the enzyme was highly susceptible to inactivation by a substance or substances present in the adrenal and removed during purification of the enzyme, and that the rate of inactivation was greatly accelerated by the presence of n- butanol at a concentration of 2%. The work reported here identified ascorbic acid as the principal inactivating factor whose action is potentiated by n- butanol . The highly purified enzyme is highly sensitive to concentrations of ascorbate several orders of magnitude lower than those found in the adrenal, and is protected from inactivation by 1 · 10 −3 M NADP + in the presence of 1% bovine serum albumin. 2. 2. Formation of hydrogen peroxide and free radicals by ascorbate appears not to be an obligatory intermediate in the inactivation process. Potentiation of ascorbate inactivation by hydrophobic alcohols and ketones is attributed to hydrophobic interactions of these substances with the enzyme. Attempts to restore activity after inactivation by ascorbate have been unsuccessful. 3. 3. The enzyme is also inactivated upon removal of NADP+ by dialysis. A 3-fold reactivation may be accomplished by incubation in the presence of NADP+ and β-mercaptoethanol.

An equation of consistency relating the harmonic mean relaxation time to sedimentation data.

Abstract 1. 1. A shape-dependent function that is independent of hydrodynamic volume has been derived and tested with data from studies of well-characterized proteins. 2. 2. The function, termed Ψ, is relatively insensitive to shape except for molecules resembling prolate ellipsoids of revolution of axial ratio > 4. 3. 3. Proposed applications of Ψ are (a) the indirect calculation of the harmonic mean relaxation time from sedimentation data for molecules of axial ratios 4, (c) testing the consistency of data from fluorescence depolarization and sedimentation data.

FLUORESCENCE QUENCHING OF 1-ANILINO-8-NAPHTHALENE SULFONIC ACID BY OXYGEN IN NORMAL ALCOHOLS

Weber and Laurence (1954) first noted that 8-anilinonaphthalene sulfonic acid (ANS), and its analogues were non-fluorescent in aqueous solutions and yet were highly fluorescent when adsorbed to certain proteins. The subsequent observation by Stryer (1965) that the fluorescence of ANS increased as the polarity of the environment decreased has led to the widespread use of ANS as a ‘hydrophobic probe’ for proteins and membranes. Although the quenching effect of oxygen on the excited state of aromatic molecules is a well known phenomenon (Wehry, 1965; Vaughn and Weber, 1970; Lakowicz and Weber, 1973), the possible role of dissolved oxygen was not discussed by Stryer (1965), and except for the recent work of Lakowicz and Weber (1973) the oxygen quenching of ANS fluorescence appears not to have been reported. In this study, we have re-examined some of Stryer’s data (1965), paying particular attention to oxygen concentration, and report measurements of the fluorescence of ANS in alcohols of varying chain length equilibrated with oxygen, nitrogen and air. As expected, the dependence of of fluorescence intensity on the amount of dissolved oxygen is almost as great as its dependence upon alcohol chain length.