Anomalous Electrophoretic Behavior Research Articles

Anomalous electrophoretic behavior of creatine kinase: A thiol-dependent isomeric system with migration subject to kinetic control

Moving boundary electrophoresis of creatine kinase in 0.1 I diethylbarbiturate buffer, pH 8.9, has yielded anomalous migration behavior that indicates intereonversion between two coexisting states of the enzyme at a rate comparable with the rate at which the two enzymic forms tend to separate by differential migration. Whereas a single, symmetrical boundary is observed in the ascending limb, distinct bimodality of the descending pattern is evident in electrophoresis of enzyme isolated from skeletal muscle of either rabbit or fish ( Mugil cephalus): pronounced changes in the nature of this bimodality with time are observed in the case of fish muscle creatine kinase. The abnormal migration behavior is eliminated by inclusion of dithiothreitol in the electrophoresis medium or by covalent modification of the enzyme with 5,5′-dithiobis(2-nitrobenzoic acid). Velocity and equilibrium sedimentation studies have been used to identify the macromolecular event as an isomerization, and studies of sulfhydryl content to implicate either reversible sulfhydryl oxidation or thiol-disulfide interchange in the isomerization mechanism.

Cetyltrimethylammonium bromide polyacrylamide gel electrophoresis: Estimation of protein subunit molecular weights using cationic detergents

Polyacrylamide gel electrophoresis in the presence of the cationic detergent, cetyltrimethylammonium bromide (CTAB), has been previously used to obtain more accurate estimates of the molecular weight of certain highly charged and membrane protein subunits that exhibit anomalous electrophoretic behavior in the presence of sodium dodecyl sulfate (SDS). The improved method reported herein is comparable to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) method in simplicity, time, and quality of gels, but the CTAB-PAGE method appears to have a wider range of application for diverse types of proteins. The technique may also be used for verification of molecular weight data and thus detection of possible anomalous results obtained using the anionic SDS-PAGE method. The described method eliminates the precipitates formed between ammonium persulfate and cationic detergents during gel polymerization and between cationic detergents and the protein dyes during staining that have complicated previous methods. The reliability of the technique is indicated by the high correlation coefficient (−0.97) between R f and molecular weight. Data are presented to indicate that the method can be used to estimate the subunit molecular weight of unknown proteins with a 95% level of confidence.

The anomalous electrophoretic behavior of the major sialoglycoprotein from the human erythrocyte.

The anomalous electrophoretic behavior of the major sialoglycoprotein from the human erythrocyte.

Adenylate kinase of human erythrocyte. Isolation and properties of the predominant inherited form.

Adenylate kinase exists in the human erythrocyte in a number of molecular forms with two allels at a single polymorphic locus coding for most of the enzyme forms. The predominant enzyme form, AK a, was purified to constant specific activity in excess of 3000 and appeared homogeneous by chromatography, electrophoresis, and ultracentrifugation. Sedimentation velocity and partial specific volume measurments of AK a yielded values of s20,w = 2.1 S and 0.722 cm-3per g. The molecular weight of the native enzyme was estimated to be 22,500 by sedimentation equilibrium and gel filtration analyses. The molecular weight of the denatured enzyme did not differ, indicating an absence of subunit structure in confirmation of genetic evidence of a single locus coding for the enzyme. The isolated enzyme demonstrated remarkable stability to denaturants (heat, guanidine HCl, urea) in the presence of appropriate stabilizing agents and could not be distinguished from rabbit muscle enzyme on this basis (as well as by a number of other kinetic and physicochemical parameters). The erythrocyte adenylate kinases have a common molecular size but differ in their charge properties. They demonstrate anomalous electrophoretic behavior, migrating anionic to hemoglobin in starch gel, yet exhibit isoelectric points considerably alkaline to hemoglobin (e.g. AK a, pI = 9.0) by isoelectric focusing.

Temperature-sensitive mutants of reovirus. IV. Evidence that anomalous electrophoretic migration behavior of certain double-stranded RNA hybrid species is mutant group-specific

Hybrid double-stranded RNA molecules that possess as their plus strand one of the messenger RNA species of certain reovirus temperature-sensitive ( ts) mutants and as their minus strand one of the minus strand species of wild-type reovirus RNA, migrate significantly more slowly when electrophoresed in polyacrylamide gels than the “homologous” or “reverse” hybrids. The extent of retardation is greater in gels containing reagents which weaken hydrogen bond formation such as urea or formamide than in gels lacking them; retardation is therefore likely to be caused by some feature of the secondary structure of these hybrids. Thirty-five ts mutants belonging to seven genome reassortment groups were examined in order to determine whether retardation of individual hybrid species is mutant group-specific, that is, whether it is attributable to the mutation that causes temperature-sensitivity. Fifty percent of group A mutants (13 of 26) yield retarded L2 hybrids (as defined above); this includes 3 of 4 mutants induced with proflavine, 4 of 10 mutants induced with N-methyl- N′-nitro- N-nitroso-guanidine, and 6 of 12 mutants induced with nitrous acid. For 11 of these mutants this was the only abnormality. Two group A mutants exhibit an additional abnormality, one in the S3 hybrid, the other in the M1 “reverse” hybrid (in which the plus strand is wild type and the minus strand is mutant). As previously reported, one of the two group D mutants and a group E mutant also exhibit two abnormalities each. These four mutants seem to be double mutants, but since their reversion frequencies are not abnormally low, only one mutation apparently causes temperature sensitivity. None of the mutants in group B, C, F, and G exhibit alterations in the electrophoretic migration rate of any hybrid species. Several spontaneous revertants that are not temperature-sensitive have been isolated from mutants yielding retarded hybrids. Some of these revertants do not yield retarded hybrids. Hybrid retardation is therefore linked to mutant phenotype. The results support the hypothesis that the mutations that cause temperature-sensitivity of group A and group E mutants are in genome RNA segment L2 and S3, respectively. Previously reported results suggest that the mutation that causes temperature-sensitivity of group D mutants is in genome RNA segment M2. Hybrid retardation therefore appears to be mutant group-specific.

The anomalous electrophoretic behavior of certain adsorbed protein derivatives.

SummaryDetermination of the electrophoretic mobility of PSL particles in increasing concentration of PLG showed an unexpectedly high positive mobility at a coverage corresponding to one monolayer. This elevated mobility was tentatively ascribed to a particular orientation of the adsorbing PLG molecules due to interaction with the PSL surface. Other surfaces examined suggest that the effect is produced by hydrophobic surfaces. The mobility changes were shown to be correlated with changes in the organ distribution of PSL particles injected into rats.It is a pleasure to acknowledge the enthusiastic technical help of Mr. Craig A. Warren. The work was supported in part by grants from The John A. Hartford Foundation, Inc., the National Institutes of Health (Grant No. AL 07161-02), and the Office of Naval Research. [Grant No. Nonr-4170 (00, NR 301-740)].

Anomalous electrophoretic behavior of purine and pyrimidine derivatives in triethylammonium buffers

Anomalous electrophoretic behavior of purine and pyrimidine derivatives in triethylammonium buffers