Herbert A. Sober

[1] Column chromatography of proteins: Substituted celluloses

Publisher Summary Chromatography of proteins on cellulose ion exchangers involves primarily the establishment of multiple electrostatic bonds between charged sites on the surface of the adsorbent and sites bearing the opposite charge on the surface of the protein molecule. The number of such bonds that can be established determines the concentration of competing ions required for the release of the bound molecule. Thus, proteins differing significantly in charge density, or in number of charges by virtue of size, may be expected to differ in their requirements for elution. Charge distribution can also be regarded as a factor. But it is the total effect of these factors that determines the affinity of the protein for the adsorbent, so a simple relation between any one of them and the chromatographic behavior of the protein in question is not always obtain. The situation is further modified by the possibility that in some cases nonelectrostatic forces might play an important role.

The effect of mercaptoethanol and urea on the molecular weight of hemoglobin

Abstract 1. 1. Unlike horse hemoglobin, human hemoglobin is not dissociated by concentrated urea, although its shape is considerably altered. 2. 2. Horse and human hemoglobins are equally dissociated by mercaptoethanol, and even further, but still equally, by a combination of mercaptoethanol and urea. 3. 3. The mechanism of mercaptoethanol action is not clear, but the data suggest that it attacks bonds other than those sensitive to urea. 4. 4. Sedimentation-diffusion measurements, in this study, indicate that dissociation of horse hemoglobin by concentrated urea is less complete than that found in previous reports. An explanation in terms of heterogeneity and a rapid dissociation-association equilibrium is suggested.

Gradient chromatography of human serum proteins and its application to the examination of “albumin” and “globulin” obtained by ammonium sulfate fractionation

Abstract A procedure for the chromatography of serum protein, employing Tris phosphate buffer in a compound gradient produced by a simple apparatus, is described. Whole serum protein of human origin, as well as “albumin” and “globulin” fractions obtained by precipitation with ammonium sulfate, were fractionated in this manner, and the chromatographic fractions were characterized by paper electrophoresis. In the subfractions of the supernatant “albumin,” small amounts of protein covering the entire globulin range of mobility were detected, because of enrichment. Protein having the electrophoretic mobility of albumin, but differing from the bulk of the albumin in chromatographic behavior, was found in the precipitated “globulin.” Siderophilin appeared in both fractions, whereas ceruloplasmin was found only in the precipitate. In general, the identifiable chromatographic components of the ammonium sulfate fractions emerged at the positions they occupied when whole serum was chromatographed. The few exceptions noted appeared to involve displacement effects rather than alteration of the protein by the ammonium sulfate.

Chromatography of proteins. III. Human, horse and dog hemoglobins on cation-exchange cellulose

Abstract Normal adult human and horse carbon monoxide hemoglobins were distinctly heterogeneous when chromatographed on cation-exchange cellulose. Under similar chromatographic conditions, normal adult dog carbon monoxide hemoglobin was essentially homogeneous. Chromatography of human or horse hemoglobin at room temperature resulted in elution at lower pH values than at 5 °. Also, increased chromatographic heterogeneity of the latter was observed. As determined by both chromatographic and sedimentation techniques, human and dog hemoglobins are relatively resistant to dissociation by 4 M urea, whereas horse hemoglobin is extensively dissociated under the same conditions.

Sedimentation behavior of bovine plasma albumin as a function of urea concentration and pH

Abstract 1. 1. Treatment of BPA with 3–5 M urea at pH 4.5 resulted in the appearance of three ultracentrifugal components. Only one component was seen below 3 M and above 6 M urea. 2. 2. Three peaks, with S 20, w values of approximately 4, 6, and 10 Svedberg units, were observed in 4 M urea over the pH range 3.4–5.3. Only one component, with a normal S 20, w of about 4, appeared at pH values of 5.5 and higher. 3. 3. A considerable increase in the intrinsic viscosity of BPA occurred at urea concentrations above 4 M , particularly in the interval between 4 and 5 M . The reduction in S 20, w at 5 M from that seen in 4 M urea was interpreted as being due to a marked increase in swelling of the albumin molecule. 4. 4. Possible mechanisms have been discussed for the aggregation of BPA in urea observed by sedimentation techniques, and conditions under which either partial or complete reversal occurred have been presented.

Computer-assisted characterization of oligoribonucleotides by their ultraviolet absorption

Abstract Spectrophotometric constants at pH 1, 7, and 12 were obtained for 34 di-, tri-, and tetranucleotides by computer-assisted examination of ultraviolet absorption spectra. The values given consist of absorption ratios at 6 wavelengths, the position of maxima and minima, hyperchromicity ratios, and an estimate of the purity of the compounds so examined.

Chromatographic Evaluation of Protein Mixtures

Publisher Summary This chapter presents chromatographic evaluation of protein mixtures. Protein and the ion exchanger are polyelectrolytes, and they are therefore capable of interacting at several points, provided intercharge distances are favorable. The adsorbed protein is more tightly bound than a singly charged substance under the same conditions; however, it can be eluted from a suitable ion exchanger if the pH is changed to reduce the number of charges on the protein or adsorbent, or if the salt concentration is raised to compete for the existing charges. A homologous series of polynucleotides emerges from a cellulosic anion exchange column in the order of increasing size. The conditions required for elution of a given molecule from a given adsorbent depend upon the number of bonds that can be formed among them. Therefore, a molecule having the same net surface charge density as another but exceeding it significantly in size, can require a stronger eluting condition because it can be capable of forming more bonds with the adsorbent.

Sedimentation behavior of horse carbon monoxide hemoglobin at low pH in the presence of mercaptoethanol and urea

Abstract 1. 1. The effect of mercaptoethanol, urea, and their combination upon the sedimentation rate of horse CO-hemoglobin has been determined at pH values of 4.0–7.2. 2. 2. Evidence is presented which supports the theory of a dissociation-association equilibrium between hemoglobin subunits.

Fractionation of trinucleotides from partial micrococcal nuclease digests of calf thymus DNA

Abstract The enzymic digest is first fractionated according to chain length by column chromatography on DEAE-Sephadex in 7 M urea at neutral pH. The trimer fraction is further resolved according to Gp content on DEAE-Sephadex in 50% MeOH and NH 4 -formate buffers at pH 5.2. The three subgroups of trinucleotides containing 0, 1, and 2 Gp residues, respectively, are then separated according to Ap, Cp, and Tp content on DEAE-cellulose in 7 M urea and 0.1 M formic acid. Among the 23 trimers so obtained, sequence isomers such as TpGpAp and ApGpTp are resolved by partition chromatography on cellulose columns with 30% NH 4 -sulfate at neutral pH.

The Preparation and Characterization of Large Oligoribonucleotides

Publisher Summary This chapter presents the preparation and characterization of large oligonucleotides. It is concerned primarily with a survey of methods for the preparation and characterization of oligomers and polymers derived from natural RNAs, such as those obtained from E . coli phages, TMV, and ribosomes. Ribonucleic acid occupies a central position in events involved in the enzymatic synthesis of proteins: (1) in the transmission of hereditary traits, (2) in mutability and, perhaps, (3) in memory. Derived oligonucleotides with chain lengths greater than 10 are of interest per se and as potential carriers of specific functions of the parent RNA. Synthetic polynucleotides of chain length 50–100 are widely used as templates for cell-free protein synthesis and large segments of natural nucleic acids have been considered to serve as templates for in vivo protein synthesis, but have not yet been chemically isolated from RNA.