Earle Stellwagen

Measurement of protein concentration with interferences optics

Abstract A convenient micro method for the measurement of protein concentration that uses an analytical ultracentrifuge as a differential refractometer is described. The number of interference fringes observed for a given protein solution is converted to mg protein/ml using an average refractive increment of 4.1 fringes/mg/ml.

Blue dextran-sepharose: an affinity column for the dinucleotide fold in proteins

A procedure is described to utilize blue dextran-Sepharose as an affinity chromatographic column specific for the super-secondary structure called the dinucleotide fold, which forms the binding sites for substrates and effectors on a wide range of proteins. The procedure can be used to identify proteins, either purified or in crude cellular extracts, that possess the dinucleotide fold and to significantly improve the purification procedures for those proteins that possess the fold.

Determining the electrophoretic mobility and translational diffusion coefficients of DNA molecules in free solution

The free solution mobility of DNA molecules of different molecular weights, the sequence dependence of the mobility, and the diffusion coefficients of small single‐ and double‐stranded DNA (ss‐ and dsDNA) molecules can be measured accurately by capillary zone electrophoresis, using coated capillaries to minimize the electroosmotic flow (EOF) of the solvent. Very small differences in mobility between various analytes can be quantified if a mobility marker is used to correct for small differences in EOF between successive experiments. Using mobility markers, the molecular weight at which the free solution mobility of dsDNA becomes independent of molecular weight is found to be ∼ 170 bp in 40 mM Tris‐acetate‐EDTA buffer. A DNA fragment containing 170 bp has a contour length of ∼ 58 nm, close to the persistence length of DNA under these buffer conditions. Hence, the approach of the free solution mobility of DNA to a plateau value may be associated with the transition from a rod‐like to a coil‐like conformation in solution. Markers have also been used to determine that the free solution mobilities of ss‐ and dsDNA oligomers are sequence‐dependent. Double‐stranded 20‐bp oligomers containing runs of three or more adenine residues in a row (A‐tracts) migrate somewhat more slowly than 20‐mers without A‐tracts, suggesting that somewhat larger numbers of counterions are condensed in the ion atmospheres of A‐tract DNAs, decreasing their net effective charge. Single‐stranded 20‐mers with symmetric sequences migrate ∼ 1% faster than their double‐stranded counterparts, and faster than single‐stranded 20‐mers containing A5‐ or T5‐tracts. Interestingly, the average mobility of two complementary single‐stranded 20‐mers is equal to the mobility of the double‐stranded oligomer formed upon annealing. Finally, the stopped migration method has been used to measure the diffusion coefficients of single‐ and double‐stranded oligomers. The diffusion coefficients of ssDNA oligomers containing 20 nucleotides are ∼ 50% larger than those of double‐stranded DNA oligomers of the same size, reflecting the greater flexibility of ssDNA molecules. The methods used to carry out these experiments are also described in detail.

Effect of the matrix on DNA electrophoretic mobility

DNA electrophoretic mobilities are highly dependent on the nature of the matrix in which the separation takes place. This review describes the effect of the matrix on DNA separations in agarose gels, polyacrylamide gels and solutions containing entangled linear polymers, correlating the electrophoretic mobilities with information obtained from other types of studies. DNA mobilities in various sieving media are determined by the interplay of three factors: the relative size of the DNA molecule with respect to the effective pore size of the matrix, the effect of the electric field on the matrix, and specific interactions of DNA with the matrix during electrophoresis.

Chromatography on immobilized reactive dyes.

Publisher Summary A reactive textile dye in solution can function as a competitive inhibitor for the substrate, coenzyme, or effector of a variety of proteins, often with an affinity greater than that exhibited by the competitive molecule. Both free and immobilized reactive dyes are available individually and as kits from a variety of suppliers at modest cost. The chemical structure of reactive dyes facilitates their rapid covalent coupling with porous matrices without recourse to prior chemistry, activation reagents, or spacer arms. The screening procedure can be completed rather expeditiously, requiring only a small amount of a crude protein extract, a general assay for protein such as the Lowry or Bradford colorimetric procedure, a specific assay for the protein of interest, and a supply of free or immobilized reactive dyes. The simplest screening procedure involves purchase of a kit of immobilized dyes currently available from at least two suppliers, Amicon and Sigma. Optimal conditions for elution of the desired protein from the positive immobilized dye should now be screened. Each screening should begin by application of sufficient crude extract to the positive immobilized dye to not to overload the column while supplying sufficient desired protein to facilitate reliable quantitative measurement upon its elution.

Probing the Electrostatic Shielding of DNA with Capillary Electrophoresis

The free solution mobility of a 20-bp double-stranded DNA oligomer has been measured in diethylmalonate (DM) and Tris-acetate buffers, with and without added NaCl or TrisCl. DM buffers have the advantage that the buffering ion is anionic, so the cation composition in the solution can be varied at will. The results indicate that the free solution mobility of DNA decreases linearly with the logarithm of ionic strength when the ionic strength is increased by increasing the buffer concentration. The mobility also decreases linearly with the logarithm of ionic strength when NaCl is added to NaDM buffer or TrisCl is added to TrisDM buffer. Nonlinear effects are observed if the counterion in the added salt differs from the counterion in the buffer. The dependence of the mobility on ionic strength cannot be predicted using the Henry, Debye-Hückel-Onsager, or Pitts equations for electrophoresis. However, the mobilities observed in all buffer and buffer/salt solutions can be predicted within approximately 20% by the Manning equation for electrophoresis, using no adjustable parameters. The results suggest that the electrostatic shielding of DNA is determined not only by the relative concentrations of the various ions in the solution, but also by their equivalent conductivities.

Proline peptide isomerization and the reactivation of denatured enzymes

Abstract The kinetics of slow phase reactivation of 11 single chain denatured enzymes containing between 6 and 28 proline residues were each found to be first-order having half-times ranging from 0.15 to 12.1 minutes, respectively, at 25 °C. The reactivation kinetics of selected enzymes are independent of solvent viscosity and give an activation energy of 19 kcal/mol. These results are consistent with the proposal that cis/trans proline isomerization in the denatured state is responsible for the slow phase of enzyme refolding/reactivation and with biosynthetic rates for enzyme production.

Monovalent Cation Size and DNA Conformational Stability

The effect of monovalent cations on the thermal stability of a small model DNA hairpin has been measured by capillary electrophoresis, using an oligomer with 16 thymine residues as an unstructured control. The melting temperature of the model hairpin increases approximately linearly with the logarithm of increasing cation concentration in solutions containing Na(+), K(+), Li(+), NH(4)(+), Tris(+), tetramethylammonium (TMA(+)), or tetraethylammonium (TEA(+)) ions, is approximately independent of cation concentration in solutions containing tetrapropylammonium (TPA(+)) ions, and decreases with the logarithm of increasing cation concentration in solutions containing tetrabutylammonium (TBA(+)) ions. At constant cation concentration, the melting temperature of the DNA model hairpin decreases in the order Li(+) ∼ Na(+) ∼ K(+) > NH(4)(+) > TMA(+) > Tris(+) > TEA(+) > TPA(+) > TBA(+). Isothermal studies indicate that the decrease in the hairpin melting temperature with increasing cation hydrophobicity is not due to saturable, site-specific binding of the cation to the random coil conformation, but to the concomitant increase in cation size with increasing hydrophobicity. Larger cations are less effective at shielding the charged phosphate residues in B-form DNA because they cannot approach the DNA backbone as closely as smaller cations. By contrast, larger cations are relatively more effective at shielding the phosphate charges in the random coil conformation, where the phosphate-phosphate distance more closely matches cation size. Hydrophobic interactions between alkylammonium ions interacting electrostatically with the phosphate residues in the coil may amplify the effect of cation size on DNA thermal stability.

Alkaline isomerization of ferricytochrome C from Euglena gracilis.

Abstract Euglena gracilis ferricytochrome c has a small absorption maximum at about 700 nm having an extinction of 850 ± 10 M−1cm−1. This absorption band is analogous to the more commonly found maximum at 695 nm which is observed in ferricytochromes from other sources and which is characteristic of ligation of methionine 80 with the heme ion. The 700 nm band disappears upon raising the pH to 11 giving a transition involving a single proton having an apparent pK of about 10. These results demonstrate that the phenolic ionization of tyrosine 67 is not required to trigger the alkaline isomerization of ferricytochromes c since Euglena cytochrome has a phenylalanine residue at position 67.

PROTEIN FOLDING OBSERVED BY CAPILLARY ELECTROPHORESIS IN ISOELECTRIC BUFFERS

Capillary zone electrophoresis measurements in acidic isoelectric buffers provide a sensitive and rapid method for comparison of the folding and stability of wild type, mutant or post-translationally modified proteins. The potential of the method is illustrated using the small globular protein cytochrome c.