On the relationship of amino acid composition to silver staining of proteins in electrophoresis gels: II. Peptide sequence analysis.

The quantification of proteins in silver-stained electrophoresis gels has been limited by the differences in "stainability" of different proteins. Despite efforts by many researchers, the precise basis of the reaction between silver reagents and polypeptides is still unclear, and, depending on the formulation, may even differ. We have tested the hypothesis that differences in stainability among proteins can be attributed to differences in di- or tripeptide composition. The results indicate that some order of protein structure other than short peptides accounts for the staining differences observed.

A system for automated DNA electrophoresis, molecular hybridization and electronic detection: II. Electronic detection.

We have designed and constructed an automated, computer-controlled, nucleic acid hybridization analysis system (Electrophoresis 1987, 8, 255-261). The system performs 9 simultaneous experiments, beginning with submarine electrophoretic separation of the restriction fragments and including microwave fixation of the separated fragments, denaturation, neutralization, prehybridization, hybridization, washing and drying. The final step is electronic detection of the hybridization pattern. The detector system consists of 90 Geiger-Mueller detectors arranged to simultaneously sample the 9 hybridizations at 10 positions each. The hybridization matrix is moved across the detectors by a robot arm in increments preprogrammed by the operator and the entire length of the matrix can be counted. The results are printed out as a plot of radioactive counts vs. distance from the origin of electrophoresis. We describe here the characteristics of the detection system.

The demonstration of alpha 1 antitrypsin in periapical lesions.

Discontinuous vertical gel electrophoresis was applied to samples of root canal aspirate, periapical blood, and control blood from twelve periapical lesions. The periapical sample from ten of the twelve lesions showed an additional band in the alpha 1 globulin area which was not present in the control. By means of Grabbar-Williams immunoelectrophoresis to specific antisera, the band was subsequently identified as alpha 1 antitrypsin. The results suggest that the regulation of proteolytic activity afforded by alpha 1 antitrypsin plays an important role in the pathogenesis of periapical lesions.

Computer applications in analysis, mapping and cataloging of proteins separated by two dimensional electrophoresis.

Two dimensional electrophoretic separation of complex mixtures of proteins can only be exploited to its fullest potential using sophisticated computerized spot detection, quantification, pattern recognition, pattern normalization, data reduction and data storage. We present a discussion of some of the technical problems and of the options available which will ultimately lead toward full computerization of the data.

Differences between human and rabbit transferrins.

Rabbit reticulocyte incorporation of iron from rabbit transferrin was independent of transferrin iron saturation but uptake from human transferrin was saturation dependent. Unlike human transferrin, rabbit transferrin does not surrender its iron from any unique preferred iron-binding site and can be described as functionally homogeneic. The two proteins also differ in their acid-base iron-binding properties. One human transferrin iron binding site retains an ability to bind iron at somewhat acid pH but this property is not shared by rabbit transferrin.

Preferential utilization in vitro of iron bound to diferric transferrin by rabbit reticulocytes.

59Fe uptake by rabbit reticulocytes from human transferrin-bound iron was studied by using transferrin solutions (35, 50, 65, 80 and 100% saturated with iron) whose only common characteristic was their content of diferric transferrin. During the early incubation period, 59Fe uptake from each preparation by reticulocytes was identical despite wide variations in amounts of total transferrin, total iron, monoferric transferrin and apotransferrin in solution. During the later phase of incubation, rate of uptake declined and was proportional to each solution's monoferric transferrin content. Uptake was also studied in a comparative experiment which used two identical, partially saturated transferrin preparations, one uniformly 59Fe-labelled and the other tracer-labelled with [59Fe]diferric transferrin. In both experiments, iron uptake by reticulocytes corresponded to utilization of a ferric ion from diferric transferrin before utilization of iron from monoferric transferrin.

Functional heterogeneity and pH-dependent dissociation properties of human transferrin.

Human diferric transferrin was partially labeled with 59Fe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte 59Fe uptake experiments with chemically labeled preparations indicated that iron bound at near neutral pH was more readily incorporated by reticulocytes than iron bound at low pH. The pH-dependent iron dissociation studies of biologically labeled transferrin solutions indicated that Fe3+, bound at the site from which the metal was initially utilized by the cells, dissociated between pH 5.8 and 7.4. In contrast, lower pH (5.2--5.8) was required to effect dissociation of iron that has remained bound to the protein after incubation with reticulocytes. These findings suggest that each human transferrin iron-binding site has different acid-base iron-binding properties which could be related to the observed heterogenic rabbit reticulocyte iron-donating properties of human transferrin and identifies that the near neutral iron-binding site initially surrenders its iron to these cells.

Failure of rabbit reticulocytes to incorporate conalbumin or lactoferrin iron.

Despite the remarkable molecular similarity of human lactoferrin and human transferrin, the results of this investigation indicate that human lactoferrin was unable to furnish rabbit reticulocytes with iron for heme synthesis. Although conalbumin closely resembles transferrin in many of its properties, conalbumin iron-binding differs from human transferrin iron-binding. There are conflicting reports in the literature regarding conalbumin's ability to furnish iron to reticulocytes. In this study, small amounts of lactoferrin or conalbumin were adsorbed to mature and immature cell surfaces but neither of these iron-binding proteins surrendered iron intracellularly to reticulocytes for heme synthesis.