Real-time measurement of solute partitioning to lipid monolayers

The interaction of a peripheral protein with a lipid-water interface can show a pronounced dependence on the composition and two-dimensional packing density of the lipids that comprise the interface. We report a novel optical method for measuring the adsorption of macromolecules, such as proteins and nucleic acids, and smaller solutes, such as drugs, to lipid monolayers at the gas-liquid interface. Using fluorescence emission from proteins and a small molecule, we demonstrate that the emissions from these solutes when in the aqueous phase and when associated with the monolayer can be temporally separated. Such separation allows measurement of the extent of solute adsorption, spectral characterization of the adsorbed solute, and characterization of lipid organization using adsorption kinetics. The method does not require, but is compatible with, the solute having different spectral properties in the bulk and surface phases. Indeed, if optical signals from adsorbed and soluble solute are the same or their relationship is known, absolute surface excess of adsorbed solute can be calculated without independent calibration. With appropriate instrumental configuration, the method should be adaptable for screening solutes for interaction with planar monolayers having both well-defined composition and adjustable lipid packing density.     

Computer-controlled direct scanning gel chromatography system

We have developed a system for direct scanning gel chromatography which is under direct control of a SYM-1 microprocessor which is in turn under control of a PDP 1104. Each scan consisting of ～200 data points of a 20-cm column may be obtained in as little as 3.5 s. Up to 100 scans may be obtained automatically without operator attention. A series of data manipulation programs have been written to allow determination of centroid migration rates, time difference chromatography calculations, etc. In conjunction with a mass transport simulation program it is possible to rapidly fit observed chromatography profiles. Use of the system to determine dispersion coefficients is described.     

Equilibrium gel permeation: a single-photon counting spectrophotometer for studies of protein interaction.

When a small column or flow cell packed with gel particles is completely saturated with a solution containing molecular species of interest, the average cross-sectional area occupied by the solute (partition cross section) is conveniently and precisely determined by direct optical scanning. For a mixture of interacting solutes this equilibrium gel permeation measurement yields the weight average of the species partition cross sections and the variation of this quantity with solute concentration permits determination of the solute interaction parameters (stoichiometry, equilibrium constants). We have developed a computer-controlled single-photon counting spectrophotometer for these measurements. The instrument exhibits high precision over a wide range of optical density. With counting times in the range of 10-1000 s the standard deviations on optical densities of protein solutions measured at 220 nm are typically 0.0006 at 1 OD, 0.002 at 2 OD, 0.005 at 4 OD. Beer's law tests show that deviations from linearity are less than these precision limits. Partition cross-section measurements for proteins can be made with an accuracy of better than 0.001 and information can be obtained with protein solutions at least as low as 1 mug/ml.     

Quantification of rotavirus-like particles by gel permeation chromatography

There is a lack of accurate and practical methods that require only small amounts of sample for quantifying virus-like particles (VLP). In this work, gel permeation (GP) HPLC was used to quantify double-layered rotavirus-like particles (dlRLP) produced in insect cells. The proposed methodology utilized two columns in series (pore sizes of 200 and 50 nm) and had a high precision (relative standard deviation below 5%). GP-HPLC not only allowed the routine quantification of dlRLP, but also of assembly intermediaries and other viral structures present in the samples. For the first time, kinetics of dlRLP accumulation could be followed. This methodology is valuable for designing new production processes and for optimizing dlRLP monitoring.     

Photoduplication and fluorescence measurement in polyacrylamide gel electrophoresis.

A simple method of photoduplication of gel electrophoresis, visualized with fluorescent reagents, is described. The procedure is convenient and rapid and requires no camera or expensive equipment. Using electrophoresis duplicating paper (Kodak), positive prints suitable for documentation or publication may be obtained. With usual photographic paper, negative prints may be obtained, allowing reliable measurement by scanning. The technique may be applied to protein or nucleic acid electrophoresis.     

Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy

We have implemented scanning fluorescence correlation spectroscopy (sFCS) for precise determination of diffusion coefficients of fluorescent molecules in solution. The measurement volume where the molecules are excited, and from which the fluorescence is detected, was scanned in a circle with radius comparable to its size at frequencies 0.5-2 kHz. The scan radius R, determined with high accuracy by careful calibration, provides the spatial measure required for the determination of the diffusion coefficient D, without the need to know the exact size of the measurement volume. The difficulties in the determination of the measurement volume size have limited the application of standard FCS with fixed measurement volume to relative measurements, where the diffusion coefficient is determined by comparison with a standard. We demonstrate, on examples of several common fluorescent dyes, that sFCS can be used to measure D with high precision without a need for a standard. The correct value of D can be determined in the presence of weak photobleaching, and when the measurement volume size is modified, indicating the robustness of the method. The applicability of the presented implementation of sFCS to biological systems in demonstrated on the measurement of the diffusion coefficient of eGFP in the cytoplasm of HeLa cells. With the help of simulations, we find the optimal value of the scan radius R for the experiment.     

Urinary protein profiling by high-performance gel permeation chromatography

We describe a new application of high-performance aqueous gel permeation chromatography for the analysis of human proteinuria. Separations of urinary proteins from normal subjects and patients with renal impairment were performed with TSK G 3000 SW columns. The effects of pH and icnic strength of the eluent on the separation of urinary proteins were investigated. Albumins were selectively separated from urine by affinity chromatography on Blue Sepharose CL-6B. According to the results of clinical investigations, urinary protein pattern derived from gel permeation chromatography revealed a good prediction of the site of renal involvement. Predominant excretion of proteins with lower molecular weight than albumin correlated with tubular damage. Albumin and higher molecular weight protein patterns were associated with glomerular disease. Absorbance measurements of the eluent at 280 nm were used for quantitative determination of total urinary protein. Gel permeation chromatography was compared to sodium dodecyl sulfate—polyacrylamide gel electrophoresis and the resulting protein patterns are in good agreement.     

High-performance liquid chromatography of proteins by gel permeation chromatography

The ability of two high-performance liquid chromatography gel permeation columns to separate proteins was evaluated. These columns gave satisfactory molecular weight separations for some, but not all, proteins tested. These results indicate that there are limitations in confidence of molecular weight determinations made by this technique.     

Purification of plant hormone extracts by gel permeation chromatography

Gel permeation chromatography on porous polystyrene beads has been adapted for the purification of plant extracts prior to analysis for plant hormones. The retention characteristics of gibberellins, indoleacetic acid, cytokinins and abscisic acid are presented along with chromatograms of some typical plant extracts.     

A simple method of analyzing profiles in time-difference direct optical scanning gel chromatography

A simple method is pressented for the analysis of time-difference, large-zone chromatography profiles, The method is derived from basic theory and is applicable to multicomponent as well as single-component systems. Simple computer simulations are used to demonstrate the inaccuracies of earlier, more empirical methods. This has been tested on several proteins using an inexpensive, semi-automated, data acquisition and control system.     

Analysis of serum iron by gel permeation high-performance liquid chromatography

A high-performance liquid chromatographic procedure for the determination of serum iron is reported. Serum iron extracted with methyl isobutyl ketone was converted to dibenzoylmethane chelate (molecular weight 725), and it was separated from excess dibenzoylmethane (molecular weight 224) by gel permeation chromatography. The chelate was determined by measuring ultraviolet absorption at 280 nm. Good reproducibility, recovery, and correlation with the conventional colorimetric method were observed.     

Molecular weight determination of peptides by high-performance gel permeation chromatography

A method for molecular weight determination of small peptides using Bio-Sil TSK 20 and Bio-Gel TSK 125 columns is described. The TSK 20 column provided a good separation of the standard peptides in the range from 1000-10,000 with an accuracy of less than 5% from the calculated regression line. Two combined TSK 125 columns allowed a reliable molecular weight determination in the range from 800 to 3500.     

Metal-binding proteins scanning and determination by combining gel electrophoresis, synchrotron radiation X-ray fluorescence and atomic spectrometry

In the present work, protein bands from in vitro embriogenic callus (Citrus sinensis L. Osbeck) were investigated using micro-synchrotron radiation X-ray fluorescence (muSR-XRF) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Metal-binding protein quantification was done after microwave oven decomposition of gel by synchrotron radiation total reflection X-ray fluorescence (SR-TXRF), flame atomic absorption spectrometry (FAAS) and flame atomic emission spectrometry (FAES). According to the analysis of the protein bands, it is possible to observe that both 81 and ca. 14 kDa proteins present different Fe signal intensity at different positions. The analysis of 53 kDa protein, showed even more interesting results. Besides Fe, the muSR-XRF experiments indicate the presence of Ca, Cu, K and Zn. Chemical elements such as Cu, K, Fe and Zn were determined by SR-TXRF, Mg by FAAS and Na by FAES. Ca was determined by SR-TXRF and FAAS only for accuracy check. In the mineralised protein bands of 81 and around 14 kDa band, only Fe was determined (105 and 21.8 microg g(-1)). For those protein bands (86-ca. 14 kDa) were determined, Ca, K, Cu and Zn in a wide concentration range (42.4-283, 2.47-96.8, 0.91-15.9 and 3.39-29.7 microg g(-1), respectively).     

Hight performance gel permeation chromatography of proteins

Proteins in the molecular weight range of 10 000–170 000 were separated by high performance gel permeation chromatography. Silica particles with 30 nm or 50 nm pores were derivatized with glycidoxy-propyltrimethoxysilane and used as support. The proteins were eluted with 50% formic acid. A protein fraction which induces endodermal and mesodermal tissues in amphibian gastrula ectoderm was purified by this method.     

Defining the factors that affect solute permeation of gap junction channels

This review focuses on the biophysical properties and structure of the pore and vestibule of homotypic gap junction channels as they relate to channel permeability and selectivity. Gap junction channels are unique in their sole role to connect the cytoplasm of two adjacent cells. In general, these channels are considered to be poorly selective, possess open probabilities approximating unity, and exhibit mean open times ranging from milliseconds to seconds. These properties suggest that such channels can function as delivery pathways from cell to cell for solutes that are significantly larger than monovalent ions. We have taken quantitative data from published works concerning unitary conductance, ion flux, and permeability for homotypic connexin 43 (Cx43), Cx40, Cx26, Cx50, and Cx37, and performed a comparative analysis of conductance and/or ion/solute flux versus diffusion coefficient. The analysis of monovalent cation flux portrays the pore as equivalent to an aqueous space where hydrogen bonding and weak interactions with binding sites dominate. For larger solutes, size, shape and charge are also significant components in determining the permeation rate. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Purification of radiolabeled and native polypeptides by gel permeation high-performance liquid chromatography

Some results and observations concerning the use of protein columns are presented. The combined use of four protein columns having different fractionation ranges together with a volatile triethylamine formate buffer allowed the sieving of various polypeptides according to their molecular weights over a range of 500 to 150,000. The addition of 4 or 6 m guanidine-HCl permitted the reduction of aggregation with no sacrifice in resolution or linearity. With that denaturant, rapid separation, and molecular weight determination in the range 500–90,000 is easily accomplished. Moreover, sample recoveries as determined with radiolabeled proteins always exceeded 70% while radioimmunoassay techniques can be directly applied to the column eluate. Applications to quick identification of natural fragments of a serine protease, tonin, analysis of maturation products of pro-opiomelanocortin in an in vitro pulse experiment and finally quantitation by radioimmunoassays of pituitary peptides and elution of their ¹²⁵I-labeled derivatives are described.