Formaldehyde Treated Gelatin as a Gel Exclusion Agent

A high molecular weight fraction of gelatin precipitated from a solution of Difco gelatin with polyethylene glycol has several favourable properties in gel exclusion chromatography. To manufacture the gel exclusion agent, the gelatin was dissolved in hot water, allowed to set in the cold and rendered insoluble by reaction with formaldehyde. When finely disrupted and used as bed material in columns this gel is capable of separating protein components from mixtures of proteins of widely different molecular weights. Favourable properties include elasticity at relatively low concentration, its availability and its inexpensive nature.     

`Tanned'' gelatin spheres and granules for exclusion chromatography

1. The preparation of tanned gelatin spheres and granules from high-molecular-weight gelatin is described. This material is comparatively hard, giving high flow rates, is insoluble in water at temperatures between 0 degrees and 100 degrees and is resistant to digestion by trypsin and chymotrypsin. The high-molecular-weight fraction of gelatin was prepared by precipitation with polyethylene glycol, and the spheres and granules prepared from this fraction were hardened and insolubilized by tanning with either formalin or chromium salts or both. 2. The spheres and granules were used successfully for the separation of protein molecules and other protein-aceous materials ranging in molecular weight from 200 to greater than 6000000. This gel exclusion material has several properties superior to those of other products used for similar purposes. Further, it was noticed that the porosity of the spheres differed considerably from that of the granules.     

Charaterization and hydrodynamic behaviour of modified gelatin: II. Characterization by high performance size exclusion chromatography — comparison with dextrans and proteins

Gelatin samples obtained by chemical modification (succinylation) are studied by SEC on silica based chromatographic supports. The influence of the pH of eluent mixtures (potassium phosphate added to NaCl) in the range 7-3.3 shows that the void volume peak (VVP) is lowered or even vanishes at pH 3.3 with the 3000 SW (TSK) gel. A process using an ultrasound treatment before injection is reported in order to determine accurately the molecular parameters of gelatin onto TSK gel with a minimal VV P. This peak is attributed to molecular aggregation of a part of the modified gelatin. After disaggregation by ultrasound or heat treatment the results are in good accordance with those obtained by other methods. It is demonstrated that with proteins and dextrans the TSK 3000 SW gel does not agree with the universal calibration curve (log[ν] · versus K_d as reported previously. A single calibration curve is obtained when the Stokes radius is plotted versus K_d. Gelatin fractions are eluted at pH 7 close to this calibration curve. This plot shows that gelatin fractions at pH 3.3 are not eluted by a pure size exclusion mechanism on 3000 SW gel. It is concluded that hydrophobic interactions between fractions of gelatin and the gel explain the high retention of these samples.     

Texture of SS Cross-linked Gelatin Gels

Relations among sensory properties, physical characteristics and the kinds of force supporting gel structure were studied by using thiolated gelatin.

Breaking stress of the gelatin gel was greatly influenced by contents of disulfide bonds in the gel. Young’s modulus of the gel was affected by its temperature. As to sensory properties of the gelatin gel, “hardness” highly correlated with Young’s modulus and “brittleness” correlated with breaking stress.

Therefore, it could be concluded that in the case of the gelatin gel, “hardness” was mainly attributed to hydrogen bonds and “brittleness” was to chemical bonds like disulfide linkages.     

Phase equilibria and mechanical properties of gel-like water-gelatin-locust bean gum systems

The establishment of phase equilibrium in aqueous gelatin-locust bean gum (LBG) systems in the process of cooling from 313 to 291 K in specific conditions, passes ahead of the gelation process(.) This allows the suggestion that macrostructure and mechanical properties of the system can be predicted on the basis of knowledge of its phase diagram, obtained for the liquid gelatin-LBG systems comprising gelatin molecular aggregates. Textural and rheological analysis of gel-like gelatin-LBG systems demonstrate the effect of two factors determining their mechanical properties and acting opposite each other when the concentration of LBG in the system increases: concentration of gelatin by LBG in the process of phase separation, and decrease in the density of the gel network.     

Removal of Proteolytic Enzymes with Aldehyde Treated Gelatin

In previous publications the use of gelatin, insolubilized with glutaraldehyde and formaldehyde, as a cation exchanger and gel exclusion agent respectively, was described^{1, 2}. This communication reports on an additional use of gelatin rendered insoluble with aldehydes.     

Isolation of similar rolipram-inhibitable cyclic-AMP-specific phosphodiesterases from rat brain and heart

A cyclic AMP phosphodiesterase form of rat brain cytosol was purified by means of affinity chromatography on an immobilized analog of the specific inhibitor rolipram, followed by an exclusion chromatography step. The resulting preparation presented two protein bands in polyacrylamide gel electrophoresis, both with phosphodiesterase activity. Kinetics of cyclic AMP hydrolysis by the purified enzyme proved of the Michaelis type, with a Km of 3 microM, while hydrolysis of cyclic GMP displayed anomalous negatively cooperative kinetics. At micromolar concentrations, this enzyme from hydrolyzed highly specifically cyclic AMP (50-fold faster than cyclic GMP). Cyclic GMP proved a poor competitor of cyclic AMP hydrolysis (Ki 1.04 mM). The neurotropic compound, rolipram, strongly inhibited the enzyme, in a competitive manner (Ki 0.9 microM). This enzyme displayed a molecular mass of around 44 kDa as determined by exclusion chromatography, but two molecular masses of 42 kDa and 89 kDa were observable by electrophoresis on a polyacrylamide gradient gel, compatible with an equilibrium between dimeric and monomeric forms. Isoelectric focusing of the preparation gave rise to two activity peaks of pI 4.8 and 6.7, with identical properties, probably representing two charge isomers of the same protein. An enzyme prepared from rat heart cytosol by the same techniques as for brain phosphodiesterase isolation shared numerous characteristics with the enzyme of cerebral origin, suggesting identity of the rolipram-sensitive form between the two tissues. Since the rolipram-sensitive form detected in crude brain preparations markedly differs from the above-described isolated enzyme, both by its molecular mass in exclusion chromatography and by its pI, it is suggested that an alteration of the native protein, due to dissociation of putative subunits, occurs during the purification procedure.     

Screening of Enterobacterial Contamination During Gelatin Production and its Effect on Pharmaceutical Grade Gelatin

Summary Gelatin, an animal protein derived from collagen has many industrial applications; mainly in food and pharmaceutical products. In this study, enterobacterial contamination of gelatin during different stages of its manufacturing was examined. Since gelatin is extracted in the form of liquor by hot water treatment of ossein and undergoes a complex series of processing stages before being finally blended and packaged off as dry gelatin product, contamination at any stage affects the quality of the final product. In total, 142 samples of gelatin were analysed for the presence of enteric bacteria, which were obtained from different stages of gelatin processing. The Enterobacteriaceae-positive samples were processed and these enterobacterial species were isolated and identified as Proteus mirabilis, Serratia marcescens, E. coli, Salmonella typhi, Klebsiella oxytoca, Klebsiella pneumoniae, Shigella flexneri and Serratia liquefaciens. These were tested for gelatinase activity. The eight species producing maximum gelatinolysis were selected and protease zymography was performed using 1% (w/v) gelatin as a substrate in 7.5% (w/v) acrylamide gel and their molecular weights were determined. The effect of these bacteria on the quality of gelatin was assessed chromatographically in terms of change in amino acid content of gelatin broth inoculated by these species, which showed a marked change with length of incubation. Since these enteric bacteria possess pathogenic properties, some of them are gelatinolytic and also have a significant effect on the quality and amino acid content of gelatin, they are of great concern both for the manufacturers as well as for the consumers.     

Purification and properties of a new type of protease produced by Microbacterium liquefaciens

A bacterium, identified as Microbacterium liquefaciens MIM-CG-9535-I, was isolated from a soil sample taken from the industrial site of a gelatin manufacturer. A new type of protease, which restrictively decomposes gelatin at one or two positions, was purified from the bacterial culture. The molecular mass of the purified enzyme was 21 kDa by SDS-polyacrylamide gel electrophoresis. The purified enzyme specifically degraded the alpha-chain of gelatin with a molecular weight of 100 kDa into two peptides of 60 kDa and 40 kDa. Native collagen was not a substrate for the enzyme.     

Isolation, purification and characterization of collagenase from hepatopancreas of the land snail Achatina fulica

Collagenase (matrix metalloproteinase-1, EC:3.4.24.7) was isolated from the hepatopancreas of Achatina fulica and characterized for its enzymatic activity and immunological properties. Procollagenase was isolated using ammonium sulphate precipitation and gel filtration, followed by purification by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid and by dialysis in neutral buffer. In the presence of SDS and beta-mercaptoethanol, the procollagenase resolved into two subunits with molecular masses of 63 and 28 kDa, respectively. The 63 kDa fragment retained its ability to bind and degrade gelatin, but the 28 kDa was inactive. Analysis by 2D gel electrophoresis revealed that the 63 kDa fragment was basic (pIs 7.6, 7.8 and 8.15), while the 28 kDa fragment was acidic (pI 4.7 and 5.1). Western blot analysis confirmed the identity of collagenase, as only matrix metalloproteinase-1 rabbit antibodies against human matrix metalloproteinase-1 (N-terminal region) recognized both the isolated procollagenase and the 63 kDa fragment.     

Purification of an active gelatinase from collagen fiber fraction of granulation tissue in rats

Gelatinase was extracted at 60 degrees C from the collagen fiber-rich fraction of granulation tissue induced by carrageenin in rats. A large part of the extracted gelatinase was unbound to Zn-chelating Sepharose. The unbound gelatinase gave a single band corresponding to a molecular mass of 57 kDa on SDS-substrate PAGE, but showed a much higher molecular mass (greater than 200 kDa) on Sephadex G-150 gel filtration. In addition, that unbound fraction contained gelatin fragments was revealed by SDS-PAGE. When the unbound fraction of Zn-chelating Sepharose was incubated at 37 degrees C, the gelatin fragments disappeared and the apparent molecular mass of gelatinase in gel filtration decreased. This gelatin degradation of the unbound fraction was enhanced by treatment with a 4-aminophenylmercuric acetate (APMA). The results suggest that the gelatinase is bound to gelatin fragments in the unbound fraction. After the treatment with APMA, the gelatinase was purified to to homogeneity; the purified gelatinase gave a single band corresponding to a molecular mass of 57 or 67 kDa on SDS-PAGE under nonreducing or reducing conditions, respectively. The purified gelatinase is a metalloproteinase, and extensively degraded gelatin, but showed no proteolytic activity toward alpha-casein or types I and IV collagens. The results suggest that the 67-kDa active gelatinase is bound to collagen fibers and plays an important role in a rapid degradation of collagen fibers in granulation tissue.     

Impact of legume seed extracts on degradation and functional properties of gelatin from unicorn leatherjacket skin

Trypsin inhibitor was extracted from the seed flour of soybean (SB; Glycine max), mung bean (MB; Vigna radiata), cowpea bean (CP; Vigna unguiculata) and adzuki bean (AB; Vigna angularis) using 0.15 M NaCl, followed by heat precipitation at 70 °C. The extract from SB showed the highest specific trypsin inhibitory activity, followed by those from MB, CP and AB, respectively. Based on inhibitory activity staining, molecular weights (MWs) of trypsin inhibitor from SB, MB, CP and AB were 20.1, 14, 10 and 13 kDa, respectively. The SB extract powder (SBEP) containing trypsin inhibitor in the range of 10–100 TIU/g effectively prevented the degradation of γ-, β- and α-chains of collagenolytic proteins of leatherjacket skin subjected to incubation at 50 °C for 30 min. The impact of SBEP on the extraction yield, physical and functional properties of gelatin from leatherjacket skin was investigated. The gelatin extracted in the presence of SBEP contained α₁ and α₂ chains as the predominant components with some degradation peptides. FTIR spectra indicated the significant loss of molecular order of triple helix and higher degradation was found in gelatin extracted in the absence of SBEP. Gelatin extracted in the presence of SBEP had the higher gel strength (232.8–268.5 g) than that extracted in the absence of SBEP (90.4 g). Higher foam stability (FS) but lower emulsion stability index (ESI) was observed in the former. Therefore, the addition of SBEP effectively prevented the degradation of gelatin from the skin of unicorn leatherjacket, thereby yielding the gelatin with improved gel strength and foam stability.     

Subunit interactions in human plasma fibronectin

The fibronectin molecule was split chemically into its two constituent chains (mol. wt. 220,000) by mild reduction with dithiothreitol. However, physical properties (molecular weight and diffusion coefficient from light scattering, and elution in gel exclusion chromatography) remained those of intact fibronectin, except (reversibly) in the presence of denaturants which also change the conformation of non-reduced fibronectin to a more open form. Similarly, during digestion of fibronectin by plasmin to fragments of molecular weight less than 200,000, the light scattering intensity drops to roughly half in 30% glycerol but not in the absence of glycerol. These results suggest that the compact conformation of native fibronectin is stabilized by specific noncovalent contacts between constituent chains.     

Biochemical characterisation of the Li locus,which controls the activity of the cyanogenic β-glucosidase in Trifolium repens L.

The cyanogenic -glucosidase (linamarase) was purified from white clover leaf tissue. The enzyme is a homodimer with a molecular weight of 105 300–103 400 daltons estimated from molecular exclusion chromatography. The effect of buffer ions on the pH optimum and charge properties of the enzyme are presented. A combination of molecular exclusion chromatography and CM cellulose ion exchange chromatography purified linamarase 16 fold to a single 62 000 dalton polypeptide on SDS polyacrylamide gel electrophoresis. This polypeptide represented about 5% of the total soluble leaf protein and can be seen as a prominent band in SDS polyacrylamide gel electrophoresis of crude leaf extracts from Li Li plants. Screening backcross progeny showed that extracts from li li plants, which have no linamarase activity, lack this 62 000 dalton polypeptide. Linamarase is the major glycoprotein in white clover leaf extracts which binds to Concanavalin A-Sepharose.     

Concentration of high molecular weight solute by swelling of hydrogel

Summary The size exclusion effect produced by the swelling of hydrolyzed polyacrylamide gel was exploited to concentrate a high molecular weight solute in batch. Changing the solvent composition induced gel swelling or shrinking. Several solutes with different molecular weights were used to test the degree of hydrogel exclusion.     

Analytical exclusion chromatography

This review summarizes the development of exclusion chromatography, also termed gel filtration, molecular-sieve chromatography and gel permeation chromatography, for the quantitative characterization of solutes and solute interactions. As well as affording a means of determining molecular mass and molecular mass distribution, the technique offers a convenient way of characterizing solute self-association and solute-ligand interactions in terms of reaction stoichiometry and equilibrium constant. The availability of molecular-sieve media with different selective porosities ensures that very little restriction is imposed on the size of solute amenable to study. Furthermore, access to a diverse array of assay procedures for monitoring the column eluate endows analytical exclusion chromatography with far greater flexibility than other techniques from the viewpoint of solute concentration range that can be examined. In addition to its widely recognized prowess as a means of solute separation and purification, exclusion chromatography thus also possesses considerable potential for investigating the functional roles of the purified solutes.     

Protein/polysaccharide cogel formation based on gelatin and chemically modified schizophyllan

In the work, aldehyde groups were quantitatively introduced into schizophyllan (SPG) side chains through periodate oxidation. The periodate-oxidized SPG (POSPG) forms an elastic gel with gelatin. The cogel formation is based on the Schiff-base reaction between the amino groups of gelatin chains and the aldehyde groups of POSPG chains. The POSPG/gelatin cogel has an elastomeric character with a very small value of loss tangent. The gelation kinetics and gel properties were discussed as a function of POSPG concentration, gelatin concentration, oxidation degree, temperature, and pH. This method can be used to design a large variety of cogels between SPG and proteins.     

FRACTIONATION OF GELATIN

1. It is possible to fractionate gelatin by means of reprecipitation at 23°C. of a salt-free solution of pH 4.7 into two fractions, one of which is soluble in water at any temperature, and a second one which does not dissolve in water even when heated to 80°C. 2. The proportion of the soluble fraction in gelatin is much greater than of the insoluble one. 3. The insoluble fraction of gelatin does not swell when mixed with water, but it does swell in the presence of acid and alkali which finally dissolve it. 4. Blocks of concentrated gel made by dissolving various mixtures of the soluble and insoluble fractions of gelatin in dilute NaOH swell differently when placed in large volumes of dilute buffer solution pH 4.7 at 5°C. The gel consisting of the insoluble material shows only a trace of swelling, while those containing a mixture of soluble and insoluble swell considerably. The swelling increases rapidly as the proportion of the soluble fraction increases. 5. A 5 per cent gel made up by dissolving the insoluble fraction of gelatin in dilute NaOH loses about 70 per cent of its weight when placed in dilute buffer pH 4.7 at 5°C. A similar gel made up of ordinary gelatin loses only about 20 per cent of its weight under the same conditions. 6. It was not found possible to resynthesize isoelectric gelatin from its components. 7. An insoluble substance similar in many respects to the one obtained by reprecipitation of gelatin is produce on partial hydrolysis of gelatin in dilute hydrochloric acid at 90°C.     

Phase equilibria and gelation in gelatin/maltodextrin systems — Part III: phase separation in mixed gels

Co-gels of potato maltodextrin Paselli SA-6 with gelatin were prepared by rapid quenching of mixed solutions from 90°C. At fixed setting temperature and fixed concentration of gelatin, the time required to form a self-supporting network showed an initial steady decrease with increasing concentration of SA-6 (as expected from polymer exclusion), but then increased dramatically before again decreasing. The interpretation of this behaviour as phase inversion from a gelatin-continuous network with SA-6 inclusions to a (more slowly-forming) SA-6 network with gelatin inclusions was confirmed by differential scanning calorimetry (showing both components melting separately, with no evidence of specific interaction), mechanical spectroscopy (showing that the mixed gel network was destroyed completely by melting of the gelatin component at low concentrations of SA-6, but only weakened at SA-6 concentrations above the inversion point) and by light microscopy (showing the expected changes in distribution of the two polymers).

In similar studies using the faster-gelling potato maltodextrin Paselli SA-2, microscopy and gel-melting profiles again showed phase-inversion from a gelatin-continuous network at low concentrations of SA-2 to a maltodextrin-continuous network at higher concentrations. Inversion, however, occurred at a lower concentration of maltodextrin than in the gelatin/SA-6 systems, and the accompanying change in gelation rate was confined to a sharp decrease in the dependence of gel-time on SA-2 concentration.     

Rheological properties of mixtures of protein-polysaccharide-dynamic viscoelasticity of blend gels of acylated gelatin, kappa-carrageenan, and agarose

Complex Young's modulus of blend gels of gelatin and kappa-carrageenan or agarose has been measured in order to clarify the protein-polysaccharide interaction in biological systems. The mixture of gelatin and kappa-carrageenan showed phase separation in the intermediate volume fraction of gelatin, and it formed a homogeneous gel when the volume fraction of gelatin is very large or very small. Since the dynamic Young's modulus for blend gels of kappa-carrageenan and gelatin was larger than the calculated one from a theory for dispersed systems, some structural reinforcing must occur. The mixture of agarose and gelatin showed the inverse tendency. It was concluded that the role of electrolytic groups was dominant in dilute gels, while molecular entanglement became more important in concentrated gels.