Cross-linked Bovine Serum Albumin Research Articles

Comparison of agarose and cross-linked protein gels as magnetic resonance imaging phantoms

Measurements of the magnetic field dependence of spin-lattice relaxation rates and the response of the water-proton signal intensity to off-resonance radio frequency fields show that the commonly used agarose phantom provides a less faithful representation for the magnetic response of tissue than does a cross-linked protein system. The origin of these differences lies in the structure and intramolecular dynamics of the macromolecular system used to make the gel. These distinctions will also cause differences in the magnetic response of the water spin system when paramagnetic relaxation agents or contrast agents are incorporated. Use of a thermally cross-linked bovine serum albumin phantom is suggested.

Post-feeding induction of trypsin in the midgut of Aedes aegypti L. (Diptera: Culicidae) is separable into two cellular phases

The induction of trypsin activity in the midgut of the mosquito, Aedes aegypti, was studied following meals of chicken blood, and several protein and peptide diets. Various concentrations of bovine serum albumin (BSA) in 0.15 M NaCl stimulated trypsin activity, in a similar fashion to the initial increase observed after a normal blood meal. Trypsin synthesis was also initiated when Ae. aegypti were fed on glutaraldehyde cross-linked BSA and on BSA fragments prepared by both pepsin and cyanogen bromide cleavage. Non-soluble proteins, in the form of glutaraldehyde-fixed erythrocyte ghosts, induced a delayed and reduced trypsin response, whilst small peptides from neutralized liver digests did not induce trypsin activity until 8–10 h after feeding. Metabolic inhibitors had varying effects on the post-feeding activity of trypsin stimulated by BSA feeding. Cycloheximide, a peptidyl transferase inhibitor prevented expression of all activity in vivo, whereas α-amanitin (RNA-polymerase inhibitor) did not affect trypsin activity in the first 10 h after feeding. At 20 μg/ml concentration in the diet, actinomycin D (RNA synthesis inhibitor) caused temporary superinduction followed by inhibition of trypsin activity, but at lower concentrations, the later phase of trypsin activity was inhibited. The results suggest that post-feeding induction of trypsin activity in Ae. aegypti is a two-phase process regulated at the midgut cellular level. The first phase of trypsin synthesis is stimulated by soluble proteins of variable molecular weights, and only involves translation of messenger RNA already available within the midgut cells. The second phase is stimulated by small peptides and requires complete synthesis of new mRNA from DNA.

Nuclear magnetic cross-relaxation spectroscopy

Magnetic cross relaxation is studied in a coupled two-spin population system. Equations for the magnetization of one spin system in the presence of partial or total saturation of the second spin system are developed. These equations predict the dependence of the signal intensity of the observed spin population on the relaxation parameters of the second coupled spin population and the frequency and amplitude of the RF field. These equations may be used to gain a qualitative understanding of the factors affecting the Z spectrum, i.e., the spectrum acquired by observing the intensity of one resonance as a function of the frequency of a saturating preparation pulse applied selectively to the other. The contributions of the different relaxation and magnetization transfer rates are analyzed analytically and demonstrated experimentally in samples of cross-linked bovine serum albumin. The data demonstrate that the predominant mechanism for the coupling is the magnetic exchange or cross relaxation between the protein and the solvent rather than the chemical exchange of exchangeable protons on the protein.

Interactions of methemoglobin and green hemoprotein in chemiluminescent gels

Peroxidation of luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) catalyzed by human green hemoprotein (GHP) and bovine methemoglobin (MetHb) in gels composed of cross-linked bovine serum albumin was examined. The chemiluminescence (CL) was followed with a low-light intensity video camera and imaging system attached to a circularly polarized microwave guide (2450 MHz) for heating the samples from 24°C to 37°C. Steady-state CL was maintained in the gels for 10 min. The intensity of the CL varied with temperature. When combined with MetHb in the same gel, GHP inhibited CL of MetHb from 83.6% to 98.2% over a fiftyfold concentration range of GHP. Although MetHb/GHP combination gels were inhibited, they generated a 6.12-fold CL per °C changes compared to a 0.19-fold per °C change for MetHb gels and a 0.31-fold per °C change for GHP gels. The data suggest an interaction between GHP and MetHb that inhibits the CL reaction, is not interfered with by large amounts of albumin, and is partially reversed by heating.

Effect of some drugs and additives on the cross-linking of bovine serum albumin by glutaraldehyde

The effect of sodium cromoglycate and sodium salicylate and a range of diverse pharmaceutical adjuvants: lactose, sodium dodecyl sulphate (SDS), sodium chloride, ammonium sulphate, including sodium benzoate and sodium salicylate as hydrotropic solubilisers, was studied on the cross-linking of bovine serum albumin (BSA) by glutaraldehyde. Photon correlation spectroscopic data indicated that the substances tested affected to different degrees the rate and extent of cross-linking, the effect being most pronounced in the presence of the inorganic salts. The results have been attributed to possible denaturation of BSA and the dehydrating properties of the inorganic salts, as both exposure of additional lysine moieties upon denaturation and dehydration are important factors in the mechanism of cross-linking. Polyacrylamide gel electrophoresis of BSA subjected to cross-linking in the presence of sodium benzoate and sodium salicylate at hydrotropic concentrations indicated a change in the nature of the cross-linked product. The possible effect of drugs and additives on the cross-linking of albumin is a factor that should be taken into consideration in the formulation of albumin microspheres as drug carriers.

Direct liquid chromatographic separation of enantiomers on immobilized protein stationary phases: VII. Sorbents obtained by entrapment of cross-linked bovine serum albumin in silica

Chiral sorbents have been prepared by physical immobilization of bovine serum albumin (BSA), cross-linked with glutaraldehyde, into silica pores. BSA i

The effect of a protein on the radiolysis of DNA studied by HPLC and pulse radiolysis.

Double-stranded DNA from calf thymus was irradiated in the presence of bovine serum albumin (BSA) with a ratio of 1:10 in weight, at pH7 and pH5, under aerobic and under anaerobic conditions. The irradiated biomolecules were separated by high-performance liquid-gel permeation chromatography. At pH 7, in the presence of the protein, degradation of DNA was enhanced by oxygen, while under anaerobic conditions formation of protein-DNA crosslinks was observed. At pH5, crosslinking of BSA to DNA occurred under anaerobic as well as under aerobic conditions, while fragmentation of DNA could not be detected with this method with doses up to 1600 Gy. Under nitrogen, the degradation of BSA was not altered by the addition of DNA, but in the presence of oxygen less BSA was lost for a given dose when DNA was present.

Penetration rate of glutaraldehyde in various buffers into plant tissue and gelatin gels

SUMMARYThe choice of buffer vehicle during fixation with glutaraldehyde influences not only the rate of crosslinking in a bovine serum albumin model system, but also the penetration rate of the glutaraldehyde into plant tissue and gelatin gels. The rate of penetration into plant tissue and the crosslinking rate of bovine serum albumin by glutaraldehyde are independent of each other in any one buffer. Buffer penetration into gelatin gels is not as fast as glutaraldehyde penetration into the same gels. The highest rate of crosslinking of bovine serum albumin by glutaraldehyde is obtained with a buffer vehicle consisting of Na2HPO4 and NaH2PO4. Glutarldehyde concentrations of 2–3% are advised for routine fixation of plant material.

Preembedding labeling with biotinylated antibodies and subsequent visualization of the biotin groups exposed on thin sections.

The feasibility of labeling cell membranes with biotinylated ligands and detecting the biotin groups on thin sections was investigated. Fixed retinal tissue was incubated with biotinyl- antiopsin . Half of the biotinyl-antibody labeled retinal tissue was incubated with avidin-ferritin (AvF) and embedded in Epon (preembedding reaction). The second half was embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Thin sections of this preparation were incubated with AvF to detect biotinyl-antibodies exposed by the sectioning (postembedding reaction). Biotin groups on the thin section surface could be readily visualized with AvF. Stereoscopic images demonstrated that the ferritin particles were localized only on the exposed surface of the thin section. The labeling was highly specific, with a very low background. Quantitative analysis was employed in order to determine the optimal reaction conditions for maximizing the labeling density with minimizing nonspecific binding. The possibility of using biotinylated molecules in the study of dynamic cellular events and for the subsequent intracellular localization of biotin on thin sections is suggested.

The interaction between collagens and factor VIII/von Willebrand factor: Investigation of the structural requirements for interaction

The blood protein Factor VIII/von Willebrand factor (FVIII/VWF) has been shown to bind to a variety of collagen polymers including (i), the native-type fibres (of collagens types I and III), (ii), segment-long-spacing (SLS) aggregates (of collagens types I, III, IV and V), (iii), the insoluble polymer obtained by random cross-linking of the type I monomer and (iv), the non-striated fibril (of type I) produced by alcohol precipitation. Relatively little binding of FVIII/VWF to the amorphous, non-fibrillar form of collagen (type I) produced by salt precipitation from acid solution was observed. No significant binding either to elastin or to the insoluble polymer derived by random cross-linking of bovine serum albumin was noted. The absorption of FVIII/VWF to collagens was affected by ionic concentration and FVIII/VWF was only totally bound at relatively low ionic strength. Binding of radiolabelled FVIII/VWF could be largely inhibited by an excess of the unlabelled protein. The interaction of FVIII/VWF with collagen fibres was inhibited in a concentration-dependent manner by monomeric collagen when present at relatively high concentrations. Gelatin did not appear to inhibit binding significantly. The structural requirements of collagen for binding to occur appear to resemble those required for collagen-induced platelet aggregation in which collagen quaternary structure rather than collagen type per se is the important factor. Loss of secondary or higher orders of structure of FVIII/VWF as a result of heat denaturation or reduction of disulphide bonds decreased or prevented binding. In accord with the association of biological activity with FVIII/VWF aggregates, optimal binding appeared to require the presence of aggregated FVIII/VWF.

On the role of cross-linking of cellular proteins in aging

Water-insoluble protein fractions increase in the brain cortical tissue and liver of rats during aging in both sexes. This suggests a possible increase in the cross-linking of proteins which may be due to the formation of, for example, hydroxyl free radicals during several metabolic processes. In vivo application of centrophenoxine causes a reversal of this phenomenon in old rats. In vitro experiments show that the generation of hydroxyl free radicals by chemical systems like homolysis of H 2O 2 by redox coupling with Fe 2+ → Fe 3+ conversion, results in the cross-linking of bovine serum albumin and the mixed proteins of liver or brain homogenates of young rats. The cross-linked proteins have a very much increased molecular weight, they become mostly insoluble even in 6 M urea. Dimethylaminoethanol, the effective part of the centrophenoxine molecule, is able to diminish the extent of cross-linking, acting most probably as a free-radical scavenger. The results are discussed in terms of the “membrane hypothesis of aging”. A molecular basis is proposed for the anti-aging effect of centrophenoxine.

Immunocytochemical localization of a large intrinsic membrane protein to the incisures and margins of frog rod outer segment disks.

Immunocytochemical techniques have localized a large protein which is an intrinsic membrane component of isolated frog rod outer segments (ROS). This large protein whose apparent mol wt is 290,000 daltons comprises about 1--3% of the ROS membrane mass. Its molar ratio to opsin is between 1:300 and 1:900. Adequate immune responses were obtained with less than 30 microgram (100 pmol) of antigen per rabbit. Antibodies to the large protein were used for its localization on thin sections of frog retina embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specifically bound antibodies were detected by an indirect sequence with ferritin-conjugated antibodies. This technique detected the protein which is represented by 1,000--3,000 molecules per disk. This indicates that the procedure is sufficiently sensitive for analysis of membrane components in low molar proportions. The large protein was specifically localized to the incisures of ROS disks which divide the disks into lobes and to the disk margin. Thus, opsin is mobile within the membrane of the disk while the large protein is apparently constrained to the disk edges. This finding raises the possibility that special functions are also localized ot his unusual region of high curvature, and that collisions of bleached opsin with these edges are physiologically important in couter segment function.

Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells. An electron microscope analysis of cross-linked albumin-embedded retinas

Adult vertebrate retinal cells (rod and cones) continuously synthesize membrane proteins and transport them to the organelle specialized for photon capture, the outer segment. The cell structures involved in the synthesis of opsin have been identified by means of immunocytochemistry at the electron microscope level. Two indirect detection systems were used: (a) rabbit antibodies to frog opsin were localized with ferritin conjugated F(ab')2 of sheep antibodies to rabbit F(ab')2 and (b) sheep antibodies to cattle opsin were coupled to biotin and visualized by means of avidin-ferritin conjugates (AvF). The reagents were applied directly to the surface of thin sections of frog retinal tissues embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specific binding of anti-opsin antibodies indicates that opsin is localized in the disks of rod outer segments (ROS), as expected, and in the Golgi zone of the rod cell inner segments. In addition, we observed quantitatively different labeling patterns of outer segments of rods and cones with each of the sera employed. These reactions may indicate immunological homology of rod and cone photopigments. Because these quantitiative variations of labeling density extend along the entire length of the outer segment, they also serve to identify the cell which has shed its disks into adjacent pigment ipithelial cell phagosomes.

A new method for the investigation of the kinetics of the capture reaction in phosphatase cytochemistry. II. Theoretical and experimental study of phosphate diffusion from thin polyacrylamide films.

Polyacrylamide films containing [32P] inorganic phosphate are proposed as models for the investigation of diffusion of phosphate from enzymatic sites. In connection with the use of such films for the study of metal salt capture reactions in phosphatase cytochemistry, the diffusion characteristics of the system were investigated both theoretically and experimentally. Diffusion in a theoretical model was simulated with aid of a computer program based on Fick's law. For films with a thickness above a certain value it proved possible to represent the progressive decrease in concentration by a hypothetical surface layer with zero concentration, the thickness being proportional to the square root of the product of the diffusion coefficient and time. The experimentally measured decrease in concentration showed similar characteristics. A slight deviation from the theoretical model indicated a retarding effect of an unstirred layer on the diffusion process. The apparent diffusion coefficient for inorganic phosphate with a pH of 5.0 at 25°C was estimated at 3.2 x 10–6 cm2 sec–1, but in the presence of 14% sucrose (w/v) this value was reduced to 2.3 x 10–6 cm2 sec–1. In films containing 6.7% w/v cross-linked bovine serum albumin Dapp was found to be 3.1 x 10–6 cm2 sec–1. This method offers a new approach to the accurate measurement of diffusion coefficients of small molecules in macromolecular matrices and is suitable for the study of diffusion problems in cytochemistry.

A method for staining intracellular antigens in thin sections with ferritin-labeled antibody.

Localization of macromolecules in biological specimens by the use of ferritin-conjugated antibody was first investigated by Singer and Schick (1961), and since that time the technique has been used to stain extraceUular antigens by several workers (Baker and Loosll, 1966; Easton et al., 1962; Morgan et al., 1961). The identification of intracellular antigens was hampered by the lack of adequate penetration of the conjugated ferritin into cells. It was therefore necessary to disrupt the cell membranes by various methods such as dissection of tissue with a razor blade (Andres et al., 1962) or by freezing and thawing (Morgan et al., 1963) to allow access of the ferritinconjugated antibody to the intraceUular antigens. A better method would be the application of the ferritin-conjugated antibody to thin sections of embedded cells so that those antigens on the surface of the sections would be able to combine with the antibody. Two main problems in applying this technique are the destruction of the antigenic determinants by the fixation and embedding and the nonspecific attachment of conjugated antibody to the surface of the embedding plastic (Striker et al., 1966). These problems have been partly overcome by McLean and Singer (1964) by the use of cross-linked polyampholyte and, more recently, by the use of cross-linked bovine serum albumin (McLean and Singer, 1970). A review of studies on the problems of staining of thin sections with the immunoferritin technique has been presented by Sternberger (1967). We report here the results of experiments dealing with the staining of intracellular antigens in thin sections of cells fixed in formaldehyde and erabedded in the water-soluble medium, glycol methacrylate (GMA). 1 In the one case the cells were adenovirus-infected cells (Shahrabadi and Yamamoto, 1970), and in the other the cells were either Micrococats sodonensis or purified cell walls of M. sodonensis.

Localization of Intracellular Antigens in thin Sections with Ferritin Labeled Antibody

The technique of labeling of macromolecules with ferritin conjugated antibody has been successfully used for extracellular antigen by means of staining the specimen with conjugate prior to fixation and embedding. However, the ideal method to determine the location of intracellular antigen would be to do the antigen-antibody reaction in thin sections. This technique contains inherent problems such as the destruction of antigenic determinants during fixation or embedding and the non-specific attachment of conjugate to the embedding media. Certain embedding media such as polyampholytes (2) or cross-linked bovine serum albumin (3) have been introduced to overcome some of these problems.

The reactions between formaldehyde, glutaraldehyde and osmium tetroxide, and their fixation effects on bovine serum albumin and on tissue blocks

The reactions between osmium tetroxide and glutaraldehyde and formaldehyde were investigated. It was found that they react together to form intermediate products which then break down to form osmium black. Glutaraldehyde reacts much more rapidly with osmium tetroxide than formaldehyde. The rates of the reactions are increased by increasing the glutaraldehyde concentration or adding bovine serum albumin to the reaction mixture. The reaction rates increase with temperature. The mixtures of fixatives were also tried on tissues and the results paralleled the model experiments. The crosslinking of bovine serum albumin by osmium tetroxide, formaldehyde and glutaraldehyde singly and in mixtures was quantitatively assessed by viscosimetry, gel filtration and disc electrophoresis coupled with densitometry. The crosslinking of bovine serum albumin by pairs of fixatives was less than that produced by the most effective of the pair. After 5 min reaction osmium tetroxide was the most effective crosslinking agent according to viscosimetric experiments, but after one hour's reaction with bovine serum albumin, glutaraldehyde was revealed as the most effective crosslinking agent by gel filtration and electrophoresis.