Hydrophobic interaction chromatography of proteins. III. Unfolding of proteins upon adsorption

Hydrophobic interaction chromatography (HIC) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. In contrast to reversed-phase chromatography, this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. Hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. When proteins are injected on HIC columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. The higher the salt concentration, the lower is the amount of eluted protein. The rest can be desorbed with a buffer of low salt concentration or water. It has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. This has been also corroborated by physicochemical measurements. The retention data of 5 different model proteins and 10 different stationary phases were evaluated. Partial unfolding of proteins upon adsorption on surfaces of HIC media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. Furthermore, we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain.     

Hydrophobic interaction chromatography of proteins on Separon hema. I. The effect of an initial salt concentration on the separation of proteins

The influence of an initial salt concentration, phi(0), on the gradient separation of proteins using hydrophobic interaction chromatography on Separon HEMA 1000 was investigated. The results obtained were compared with the retention times and peak widths calculated according to a mathematical model.     

Microemulsion electrokinetic chromatography of proteins.

Microemulsion electrokinetic chromatography was used to separate a test mixture of proteins effectively. The separation was carried out in a 42.5 cm (to the detector) x 50 microns I.D. fused-silica capillary using a microemulsion system consisting of 80 mM heptane, 120 mM SDS, 900 mM butanol in 2.5 mM borate buffer, pH 8.5-9.5. Optimum separation conditions were investigated with respect to the running voltage, temperature, pH and the composition of microemulsion. Results were compared with those obtained in micellar electrokinetic chromatography and capillary zone electrophoresis. The examined method is practical and successfully applied to the assay of genetically engineering pharmaceuticals, recombinant human granulocyte macrophage colony stimulating factor injection and recombinant human granulocyte colony stimulating factor injection.     

Third-generation biosensors based on the direct electron transfer of proteins.

Recent progress in third-generation electrochemical biosensors based on the direct electron transfer of proteins is reviewed. The development of three generations of electrochemical biosensors is also simply addressed. Special attention is paid to protein-film voltammetry, which is a powerful way to obtain the direct electron transfer of proteins. Research activities on various kinds of biosensors are discussed according to the proteins (enzymes) used in the specific work.     

Detection of proteins after immunoblotting on nitrocellulose using fluorescent antibodies.

A Western blot method is described utilizing 5-dimethylaminonaphthalene-1-sulfonyl (dansyl chloride)-conjugated antibodies to detect proteins transferred onto nitrocellulose. Four proteins of different molecular weight, including human albumin, gamma heavy chain, kappa and lambda light chains of immunoglobulins, were tested. Labeling with the fluorochrome was compared with the method of enzyme-conjugated antibodies and was found to have similar sensitivity, specificity, and safety. Moreover, this method is less expensive, and the labeled antibodies can be prepared fresh in a short time and are free from the disadvantages of enzyme label whose activity is affected by the presence of activators or inhibitors in the tested specimens.     

Spectroscopy of maize proteins II. Albumins

The albumins of maize grain isolated by precipitation from a total salt extract have been investigated by IR, UV, derivative, and fluorescence spectroscopy. It has been shown that the variability of the spectral parameters is connected with a change in the association of the proteins with nonprotein substances according to the genotype of the maize. Scientific-Research Institute of Biology, Dnepropetrovsk State University. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 549–553, July–August, 1991.     

Spectroscopy of maize proteins II. Albumins

The albumins of maize grain isolated by precipitation from a total salt extract have been investigated by IR, UV, derivative, and fluorescence spectroscopy. It has been shown that the variability of the spectral parameters is connected with a change in the association of the proteins with nonprotein substances according to the genotype of the maize.Scientific-Research Institute of Biology, Dnepropetrovsk State University. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 549–553, July–August, 1991.     

Thin-layer gel chromatography of dyed proteins.

Plasma chromatography as a method for ultratrace qualitative and quantitative detection of organic compounds is especially well suited for detection of gas chromatographic effluents. The optimum range of sample quantity is 10(-6) to 10(-12) g for detection and identification of a compound by use of its characteristic positive and negative mobility spectra. The type of reference mobility spectra produced by alkanes, aromatics, esters, halogenated compounds, nitrogenated compounds and organic acids have been previously reported. This study presents the reference mobility spectra produced for lysergic acid diethylamide (LSD), delta9-tetrahydrocannabinol (delta9-THC), digitoxigenin and several biochemical compounds of research significance. LSD and delta9-THC in a mixture can be detected and identified by plasma chromatography positive mobility spectra in quantities of 10(-7) g or less. All the compounds investigated in this study display strong MH+ ions along with other ions primarily of the type (M)NO+, (M)2H+. None of these compounds exhibits negative mobility spectra.     

Characterization of proteins by mass spectrometry. Invited lecture.

Plasma desorption and fast atom bombardment mass spectrometry have in the last decade demonstrated the potential of mass spectrometry for protein studies. The recently developed matrix-assisted laser desorption and electrospray mass spectrometry have expanded the analytical potential of mass spectrometry to cover nearly all proteins. The type of information obtained with the four methods is described and their performances are compared. The potential of combining mass spectrometric relative molecular mass information on proteins with the information contained in protein sequence databases is outlined and some typical fields of application of mass spectrometry in protein chemistry are described. The need for the full integration of mass spectrometry in the protein laboratory is discussed.     

Hydrogel microspheres III. Temperature-dependent adsorption of proteins on poly-N-isopropylacrylamide hydrogel microspheres

Precipitation polymerization of N-isopropylacrylamide (NIPAM) with methylenebisacrylamide (MBAAm) in water at 70°C gave thermosensitive hydrogel microspheres. The adsorbability of proteins on the poly-NIPAM microspheres was found to depend on temperature. Below the lower critical solution temperature (LCST) of poly-NIPAM in an aqueous medium, that is, around 32°C, the microspheres hold a large amount of water inside and their surface is hydrophilic enough to suppress the adsorption of proteins. On the contrary, above 32°C, the micropheres deswell and their surface becomes hydrophobic and, consequently, susceptible to adsorption of a large amount of proteins. Proteins once adsorbed on the microspheres at a high temperature could be desorbed more or less by lowering the temperature to below 32°C. The extent of desorption at low temperatures was found to depend on the incubation time for adsorption at high temperatures.     

Structural rules for globular proteins.

Is it possible to reach a detailed understanding of the complex three-dimensional structures of native polypeptide chains? In view of the wealth of common physicochemical and phylogenetic features discovered among proteins this question has become reasonable. The current state of discussion is presented in this report.     

Immunization procedures forE. coli proteins

Three immunization procedures were compared for the production of antibodies to the minor components of a complex E. coli protein (ECP) mixture: a conventional protocol and two methods that allow for the selective in vitro (cascade) or in vivo (passive) depletion of highly immunogenic proteins. An indirect ELISA showed that a maximum ELISA antibody titer was obtained with all the procedures 60 d after immunization. Analysis of these antisera by two-dimensional SDS-PAGE immunoblots, however, demonstrated that antibody reactivity to minor components in the mixture was not achieved until 112 d. This analysis also showed that a marked improvement in antibody response to minor components was obtained with the cascade immunization procedure. The mean titer and spectrum of antibody reactivity was similar for each group, and suggested that, although some individual variation was noted, the improvements observed were the result of the protocol used. Thus, for these ECPs, and multiple antigen mixtures in general, the preferred immunization protocol should employ at least three hosts and utilize the cascade immunization of Thalhamer and Freund. Characterization of the resulting antisera is best performed by use of silver stained two-dimensional SDS-PAGE and immunoblotting.     

Cascade-mode multiaffinity chromatography. Fractionation of human serum proteins.

The group-resolving power of cascade-mode multiaffinity column chromatography (CASMAC), was demonstrated with human serum as a model mixture. More than 99% of the serum proteins were adsorbed in the same high salt-containing buffer on a tandem column consisting of (1) immobilized Zn2+ on triscarboxymethyl diamine gel followed by (2) thiophilic (T) gel, (3) Zn2+ bound to the new tridentate chelating adsorbent dipicolylamine (DPA) agarose, (4) hexyl-thioether C6-S agarose and (5) Ni(2+)-DPA agarose. After the adsorption step the immobilized metal ion affinity gels were attached to the top of tandem columns of other adsorbents (T gel, Sephadex G-25 for desalting and Mono-Q) and the elution conditions were selected such that further group separation was achieved. High resolution, high recovery, easy manipulation and high capacity are characteristic features of the cascade process with these adsorbents. The advantage of CASMAC is particularly striking when, with a given number of adsorbents, the overall number of operations involving adsorption, desorption, washing, buffer change and substance concentration can be effectively minimized.