Determination of interchain crosslinkages in insulin B-chain dimers by fast atom bombardment mass spectrometry

New methodology for identifying and locating crosslinkages in peptides is described. Pepsin is used to cleave insulin and B-chain dimers of insulin into fragments under conditions which retain the original peptide crosslinkages. After partial separation by HPLC, the peptides are analyzed by fast atom bombardment mass spectrometry (FABMS) to determine their molecular weights. The molecular weights of peptide fragments expected from the pepsin digests of human insulin and related model compounds are calculated from the amino acid sequence of the intact peptide. Digestion products are identified by matching their molecular weights, as determined by FABMS, with calculated molecular weights. Locations of interchain crosslinkages are deduced after the peptide fragments have been assigned to specific segments of the parent peptide. The validity of the method has been established by correctly identifying structurally important products in the pepsin digests of model compounds such as human, bovine, and porcine insulins. Procedures developed with the model compounds were used to identify crosslinkages in peptides of unknown structure isolated from an insulin A-chain/B-chain combination reaction mixture. Evidence is presented for formation of three different types of crosslinkages, disulfide, lanthionine, and sulfoxide.     

Phase equilibria and gelation in gelatin/maltodextrin systems — Part IV: composition-dependence of mixed-gel moduli

The storage moduli (G′) of phase-separated co-gels formed by quench-cooling mixed solutions of gelatin and potato maltodextrin (Paselli SA-2 and SA-6) have been related quantitatively to the experimentally-determined concentration-dependence of G′ for the constituent polymers. Distribution of water between the phases was examined explicitly by using polymer blending laws to derive calculated moduli for gelatin-continuous and maltodextrin-continuous networks over the entire range of solvent partition. Allowance was made for the direct contribution of polymer chains, and for density differences between the phases, in calculating relative phase-volumes. The effect of gel formation prior to phase-separation was calculated using classical theory for network de-swelling. Good agreement with observed moduli for more than 30 gelatin/maltodextrin combinations was achieved using a single adjustable parameter, p (the ratio of solvent to polymer in the gelatin phase divided by the same ratio for the maltodextrin phase), with an optimum value of p ≈ 1·8 for both SA-6 and SA-2.     

Synthesis and optimization of gelatin nanoparticles using the miniemulsion process

A convenient synthetic route based on the concept of nanoreactors using the versatility of the miniemulsion technique to synthesize glutardialdehyde cross-linked gelatin nanoparticles with tailored properties is reported. It is demonstrated that, independent of the molecular weight distribution of the gelatin used, stable nanoparticles can be produced with a small amount of surfactant. The amount of gelatin and the cross-linking degree in the particle can be well controlled. Different types of gelatin have been used without purification or fractionation. The stability of the dispersion, particle size, and the efficiency of cross-linking have been studied. Such nanoparticles with varying gelatin concentration and cross-linking density have high potential to be used for drug delivery applications, as nanoenvironment or template for synthesizing inorganic materials.     

A study of collagen and gelatin solutions by optical self beat spectroscopy

A study of the conformation of collagen and gelatin in aqueous solution by Optical Self Beat Spectroscopy is reported. The translational diffusion coefficient of monomeric tropocollagen was experimentally measured from the half-width of the Rayleigh scattered radiation and the value obtained is shown to be in good agreement with that calculated from hydrodynamical theory for the tropocollagen rod. The Self Beat Spectrometer was also used to investigate the factors affecting the aggregation and flexing of molecules in dilute gelatin solutions and the gel-sol transition in more concentrated gelatin solutions.     

HYDRATION OF GELATIN IN SOLUTION

1. It was shown that the high viscosity of gelatin solutions as well as the character of the osmotic pressure-concentration curves indicates that gelatin is hydrated even at temperatures as high as 50°C. 2. The degree of hydration of gelatin was determined by means of viscosity measurements through the application of the formula See PDF for Equation. 3. When the concentration of gelatin was corrected for the volume of water of hydration as obtained from the viscosity measurements, the relation between the osmotic pressure of various concentrations of gelatin and the corrected concentrations became linear, thus making it possible to determine the apparent molecular weight of gelatin through the application of van''t Hoff''s law. The molecular weight of gelatin at 35°C. proved to be 61,500. 4. A study was made of the mechanism of hydration of gelatin and it was shown that the experimental data agree with the theory that the hydration of gelatin is a pure osmotic pressure phenomenon brought about by the presence in gelatin of a number of insoluble micellæ containing a definite amount of a soluble ingredient of gelatin. As long as there is a difference in the osmotic pressure between the inside of the micellæ and the outside gelatin solution the micellæ swell until an equilibrium is established at which the osmotic pressure inside of the micellæ is balanced by the total osmotic pressure of the gelatin solution and by the elasticity pressure of the micellæ. 5. On addition of HCl to isoelectric gelatin the total activity of ions inside of the micellæ is greater than in the outside solution due to a greater concentration of protein in the micellæ. This brings about a further swelling of the micellæ until a Donnan equilibrium is established in the ion distribution accompanied by an equilibrium in the osmotic pressure. Through the application of the theory developed here it was possible actually to calculate the osmotic pressure difference between the inside of the micellæ and the outside solution which was brought about by the difference in the ion distribution. 6. According to the same theory the effect of pH on viscosity of gelatin should diminish with increase in concentration of gelatin, since the difference in the concentration of the protein inside and outside of the micellæ also decreases. This was confirmed experimentally. At concentrations above 8 gm. per 100 gm. of H₂O there is very little difference in the viscosity of gelatin of various pH as compared with that of isoelectric gelatin.     

Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections

We used a model system to study whether measurements of absolute local antigen concentrations at the electron microscopic level are feasible by counting immunogold labeling density in ultra-thin sections. The model system consisted of a matrix of a variable concentration of gelatin, which was mixed with given concentrations of rat pancreas amylase and fixed according to various fixation protocols. With a relatively mild fixation, there was no clear proportionality between anti-amylase gold labeling and amylase concentration in ultra-thin cryosections. This was presumably due to uncontrolled loss of amylase from the sections. After stronger fixation with 2% glutaraldehyde for 4 hr, labeling density reflected the amylase concentration very well. We observed that matrix (gelatin) density influenced labeling density. A low gelatin concentration of 5% allowed penetration of immunoreagents into the cryosection, resulting in a high and variable labeling density. In gelatin concentrations of 10% and 20%, labeling density was lower but proportional to amylase concentration. To establish an equal (minimal) penetration of immunoreagents, we embedded model blocks with different matrix densities in polyacrylamide (PAA). In ultra-thin cryosections of these PAA-embedded blocks, anti-amylase labeling was proportional to amylase concentration even at a low (5%) gelatin concentration. Anti-amylase labeling in ultra-thin sections from Lowicryl K4M low temperature-embedded blocks was higher than in PAA sections, but the results were less consistent and depended to some extent on matrix density. These results, together with the earlier observation that acrylamide completely penetrates intracellular compartments (Slot JW, Geuze HJ: Biol Cell 44:325, 1982), demonstrate that it is possible to measure true intracellular concentrations of soluble proteins in situ using ultra-thin cryosections of PAA-embedded tissue.     

THE SWELLING OF GELATIN AND THE VOLUME OF SURROUNDING SOLUTION

The swelling of isoelectric gelatin added to various volumes of acid of different concentration at 5°C. has been determined. The swelling is determined only by the concentration of the supernatant solution at equilibrium and is independent of the volume of acid. Similar experiments with unpurified gelatin show that in this case, owing to the presence of neutral salts the swelling is a function of the volume as well as the concentration of acid. Both results are predicted by the Procter-Wilson-Loeb theory of the swelling of gelatin.     

Analyzing the hydrodynamic and crowding evolution of aqueous hydroxyapatite‐gelatin networks: Digging deeper into bone scaffold design variables

The hydration of the polypeptide network is a determinant factor to be controlled on behalf of the design of precise functional tissue scaffolding. Here we present an exhaustive study of the hydrodynamic and crowding evolution of aqueous gelatin‐hydroxyapatite systems with the aim of increasing the knowledge about the biomimesis of collagen mineralization; and how it can be manipulated for the preparation of collagenous derived frameworks with specific morphological characteristics. The solution's density and viscosity evaluation measurements in combination with spectroscopic techniques revealed that there is a progressive association of protein chain that can be influenced by the amount of hydroxyapatite nanorods. Gelatin and additives’ concentration effect on the morphology of the gelatin scaffolds was investigated. Transverse and longitudinal sections of the obtained scaffolds were taken and analyzed using optical microscopy. It can be seen that the porous size and shape of gelatin assemblies can be easily adjusted by controlling the gelatin/HAp ratio in the solution used as template in agreement with our statement. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 393–405, 2015.     

1H n.m.r. study of geletin gels

Proton spin-lattice and spin-spin relaxation times have been measured in the gelatin-water system during gelatin at various concentrations and quenching temperatures. The spin-spin relaxation time is shown to be a good parameter for following the kinetics of gelation and can be related to the amount of helices measured by polarimetry. The results are analysed within the framework of the Zimmerman-Brittin theory.     

ON THE INFLUENCE OF AGGREGATES ON THE MEMBRANE POTENTIALS AND THE OSMOTIC PRESSURE OF PROTEIN SOLUTIONS

1. It is shown that when part of the gelatin in a solution of gelatin chloride is replaced by particles of powdered gelatin (without change of pH) the membrane potential of the solution is influenced comparatively little. 2. A measurement of the hydrogen ion concentration of the gelatin chloride solution and the outside aqueous solution with which the gelatin solution is in osmotic equilibrium, shows that the membrane potential can be calculated from this difference of hydrogen ion concentration with an accuracy of half a millivolt. This proves that the membrane potential is due to the establishment of a membrane equilibrium and that the powdered particles participate in this membrane equilibrium. 3. It is shown that a Donnan equilibrium is established between powdered particles of gelatin chloride and not too strong a solution of gelatin chloride. This is due to the fact that the powdered gelatin particles may be considered as a solid solution of gelatin with a higher concentration than that of the weak gelatin solution in which they are suspended. It follows from the theory of membrane equilibria that this difference in concentration of protein ions must give rise to potential differences between the solid particles and the weaker gelatin solution. 4. The writer had shown previously that when the gelatin in a solution of gelatin chloride is replaced by powdered gelatin (without a change in pH), the osmotic pressure of the solution is lowered the more the more dissolved gelatin is replaced by powdered gelatin. It is therefore obvious that the powdered particles of gelatin do not participate in the osmotic pressure of the solution in spite of the fact that they participate in the establishment of the Donnan equilibrium and in the membrane potentials. 5. This paradoxical phenomenon finds its explanation in the fact that as a consequence of the participation of each particle in the Donnan equilibrium, a special osmotic pressure is set up in each individual particle of powdered gelatin which leads to a swelling of that particle, and this osmotic pressure is measured by the increase in the cohesion pressure of the powdered particles required to balance the osmotic pressure inside each particle. 6. In a mixture of protein in solution and powdered protein (or protein micellæ) we have therefore two kinds of osmotic pressure, the hydrostatic pressure of the protein which is in true solution, and the cohesion pressure of the aggregates. Since only the former is noticeable in the hydrostatic pressure which serves as a measure of the osmotic pressure of a solution, it is clear why the osmotic pressure of a protein solution must be diminished when part of the protein in true solution is replaced by aggregates.     

Dynamic mechanical and rheo-optical studies of collagen and gelatin

The frequency dependence of dynamic mechanical properties of rat tail tendon (RTT), enzyme-solubilized collagen membranes (ESC), AKM-23 dialysis membranes, and gelatin film have been measured at 110, 11, and 3.5 Hz from - 160 to 220°C. RTT and AKM-23 are devoid of a rubbery region; there are as many as six mechanical loss transitions. Gelatin and ESC membranes behave as rubbery materials above room temperature; only three tan δE peaks can be resolved for these materials. Strain birefringence was used to measure the crystalline and amorphous contribution of orientation induced by strain. Both the birefringence and the strain optical coefficient are sensitive to the amount of water in a sample. The effect of chemical swelling agents and of annealing on birefringence are described. Stress relaxation data on gelatin film were analyzed with the rubber elasticity theory to give the average number of chains per unit volume, the specific polarizability, the stress-birefringence ratio and the average molecular weight between hydrogen bonds were calculated. The intrinsic amorphous birefringence for “wet” gelatin film is 1.25 × 10^?2; it is estimated to be about 6 × 10^?2 for “dry” gelatin film.     

Swelling and de-swelling kinetics of gelatin hydrogels in ethanol-water marginal solvent

Controlled osmotic swelling and de-swelling measurements have been performed on gelatin, a polyampholyte, hydrogels suspended in water-ethanol marginal solvent at room temperature (20 degrees C) where the alcohol concentration was changed from 0 to 100% (v/v). The change in gel mass was monitored as function of time until osmotic equilibrium was established with the surrounding solvent. It was observed that osmotic pressure of polymer-solvent mixing, pi(m)相似文献   

HeLa cell adhesion to microcarriers in high shear conditions: evidence for membrane receptors for collagen but not laminin or fibronectin

HeLa-S3 cells were analyzed for their ability to attach and spread on cell culture microcarriers that were made either positively or negatively charged with polymeric plastics or were coated with BSA, gelatin, fibronectin or laminin. The cells stuck to all microcarriers under low shear, i.e. low stirring conditions with similar rates of attachment. Except in the case of gelatin microcarriers where cells fully spread, cells did not or only partially spread on the others. Under high shear, cells attached with the following rates: positive = negative = gelatin = BSA greater than laminin greater than fibronectin. Cells detached from all but the gelatin and BSA coated beads. However, the cells did not fully spread on BSA beads. The observation that cells not only attached but also spread on gelatin beads indicated that gelatin could be a specific substratum adhesion protein while the other surfaces were 'non-specific'. It should be noted that neither antibodies to laminin nor fibronectin interfered with attachment to gelatin. Protein synthesis inhibitors reduced the attachment and spreading on gelatin beads under high but not low shear conditions. With low shear, attachment and spreading appeared normal. We concluded that the density of the cell surface attachment proteins was reduced by the protein synthesis inhibitors and there were not enough present to facilitate attachment under high shear. The results also indicated that protein synthesis was not essential for cell spreading. Proteolysis of the cell surface with low concentrations of trypsin abolished the attachment of cells to gelatin-coated beads. The reappearance of attachment ability took several hours and was inhibited by actinomycin-D.     

Study of the near-neutral pH-sensitivity of chitosan/gelatin hydrogels by turbidimetry and microcantilever deflection

The fundamental properties and pH-sensitivity of chitosan/gelating hydrogels were investigated using spectroscopic and microelectro mechanical (MEMS) measurement approaches. Turbidimetric titration revealed that there were electrostatic attractive interactions between tripolyphosphate (TPP), chitosan, and gelatin in the acidic pH range, depending on their degree of ionization. The pH-sensitive swelling behavior of the hydrogels was investigated by monitoring the deflection of hydrogel-coated microcantilevers, which exhibited a sensitive and repeatable response to solution pH. The deflection of the microcantilever increased as the pH decreased, and the response speed of the system exhibited a nearly linear relationship with pH. The effects of the pH and concentration of TPP solution, as well as the ratio of chitosan to gelatin in gel precursor solutions, on the pH sensitivity of the hydrogels were also investigated. It was found that the swelling of the hydrogel is mainly a result of chain relaxation of chitosan-TPP complexes caused by protonation of free amino groups in chitosan, which depends on the crosslinking density set during the formation of the network. An increase in initial crosslink density induced a decrease in swelling and pH sensitivity. It can be concluded from this study that pH-sensitive chitosan gel properties can be tuned by preparatory conditions and inclusion of gelatin. Furthermore, microcantilevers can be used as a platform for gaining increased understanding of environmentally sensitive polymers.     

THE ADSORPTION OF GELATIN BY COLLODION MEMBRANES

An experimental study has been made of the adsorption of gelatin from solution at 37°C. by collodion membranes. In the case of membranes of high permeability, very high concentrations of gelatin were required to produce maximum adsorption, and the maximum amounts adsorbed were independent of the pH values of the solutions over the range 3.8 to 4.8. With membranes of low permeability, maximum adsorption was reached at lower gelatin concentrations, and the maximum amounts adsorbed varied with the pH, being lower on either side of the isoelectric point, over the range 3.8 to 6.6. The addition of salt in such experiments raised the maximum amount adsorbed to a value equal to that obtained with solutions at the isoelectric point in the absence of salt. These experiments can be explained by, and seem to lend support to, the theory proposed by Loeb and further developed by Kunitz concerning the effects of pH and salt on the size of gelatin particles in solution.     

A microbial biosensor for hydrogen sulfide monitoring based on potentiometry

In this study, a microbial biosensor for hydrogen sulfide (H₂S) detection based on Thiobacillus thioparus immobilized in a gelatin matrix was developed. The T. thioparus was immobilized via either surface adsorption on the gelatin matrix or entrapment in the matrix. The bacterial and gelatin concentration in the support were then varied in order to optimize the sensor response time and detection limit for both methods. The optimization was conducted by a statistical analysis of the measured biosensor response with various bacterial and polymer concentrations. According to our experiments with both immobilization methods, the optimized conditions for the entrapment method were found to be a gelatin concentration of 1% and an optical density of 82. For the surface adsorption method, 0.6% gelatin and an optical density of 88 were selected as the optimal conditions. A statistical model was developed based on the extent of the biosensor response in both methods of immobilization. The maximum change in the potential of the solution was 23.16 mV for the entrapment method and 34.34 mV for the surface absorption method. The response times for the entrapment and adsorption methods were 160 s and 105 s, respectively. The adsorption method is more advantageous for the development of a gas biosensor due to its shorter response time.     

Influence of Probe Flexibility and Gelatin Embedding on Neuronal Density and Glial Responses to Brain Implants

To develop long-term high quality communication between brain and computer, a key issue is how to reduce the adverse foreign body responses. Here, the impact of probe flexibility and gelatine embedding on long-term (6w) tissue responses, was analyzed. Probes of same polymer material, size and shape, flexible mainly in one direction, were implanted in rat cerebral cortex (n_implants = 3 x 8) in two orientations with respect to the major movement direction of the brain relative to the skull: parallel to (flex mode) or transverse to (rigid mode). Flex mode implants were either embedded in gelatin or non-embedded. Neurons, activated microglia and astrocytes were visualized using immunohistochemistry. The astrocytic reactivity, but not microglial response, was significantly lower to probes implanted in flex mode as compared to rigid mode. The microglial response, but not astrocytic reactivity, was significantly smaller to gelatin embedded probes (flex mode) than non-embedded. Interestingly, the neuronal density was preserved in the inner zone surrounding gelatin embedded probes. This contrasts to the common reports of reduced neuronal density close to implanted probes. In conclusion, sheer stress appears to be an important factor for astrocytic reactivity to implanted probes. Moreover, gelatin embedding can improve the neuronal density and reduce the microglial response close to the probe.     

THE COLLOIDAL BEHAVIOR OF SERUM GLOBULIN

1. The globulin prepared from ox serum by dilution and precipitation with carbon dioxide has been found, by electrometric titration experiments, to behave like an amphoteric electrolyte, reacting stoichiometrically with acids and bases. 2. The potential difference developed between a solution of globulin chloride, phosphate, or acetate and a solution of the corresponding acid, free from protein, separated from the globulin by a collodion membrane, was found to be influenced by hydrogen ion concentration and salt concentration in the way predicted by Donnan''s theory of membrane equilibrium. In experiments with sodium globulinate and sodium hydroxide it was found that the potential difference could be similarly explained. 3. The osmotic pressure of such solutions could be qualitatively accounted for by the Donnan theory, but exhibited a discrepancy which is explicable by analogy with certain experiments of Loeb on gelatin. 4. The application of Loeb''s theory of colloidal behavior, which had previously been found to hold in the case of gelatin, casein, egg albumin, and edestin, has thus been extended to another protein, serum globulin.     

PCL film surfaces conjugated with P(DMAEMA)/gelatin complexes for improving cell immobilization and gene transfection

Successful gene transfection on a tissue scaffold is of crucial importance in facilitating tissue repair and regeneration by enabling the localized production of therapeutic drugs. Polycaprolactone (PCL) has been widely adopted as a scaffold biomaterial, but its unfavorable cell-adhesion property needs to be improved. In this work, the PCL film surface was conjugated with poly((2-dimethyl amino)ethyl methacrylate) (P(DMAEMA))/gelatin complexes via surface-initiated atom transfer radical polymerization (ATRP) for improving cell immobilization and subsequent gene transfection. A simple aminolysis-based method was first used for the covalent immobilization of ATRP initiators on the PCL film. Well-defined P(DMAEMA) brushes were subsequently prepared via surface-initiated ATRP from the initiator-functionalized PCL surfaces. The P(DMAEMA) chains with a pK(a) of 7.0-7.3 were used for conjugating gelatin with a pI of 4.7 via electrostatic interaction. The amount of complexed gelatin increased as that of the grafted P(DMAEMA) layer. The cell-adhesion property on the functionalized PCL surface could be controlled by adjusting the ratio of P(DMAEMA)/gelatin. It was found that the gene transfection property on the immobilized cells was dependent on the density of the immobilized cells on the functionalized PCL film. With the good cell-adhesive nature of gelatin and the efficient gene transfection on the dense immobilized cells, the incorporating the suitable of P(DMAEMA)/gelatin complexes onto PCL surfaces could endow the PCL substrates new and interesting properties for potential tissue engineering applications.     

Preparation and characterization of novel chitosan/gelatin membranes using chitosan hydrogel

Chitin and chitosan are novel biomaterials. The novel chitosan/gelatin membranes were prepared using the suspension of chitosan hydrogel mixed with gelatin. The prepared chitosan/gelatin membranes were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), mechanical, swelling, and thermal studies. The morphology of these chitosan/gelatin membranes was found to be very smooth and homogeneous. The XRD studies showed that the chitosan/gelatin membranes have good compatibility and interaction between the chitosan and gelatin. The stress and elongation of chitosan/gelatin membranes on wet condition showed excellent when the mixture ratio of gelatin was 0.50. The prepared chitosan/gelatin membranes showed good swelling, mechanical and thermal properties. Cell adhesion studies were also carried out using human MG-63 osteoblast-like cells. The cells incubated with chitosan/gelatin membranes for 24 h were capable of forming cell adhesion. Thus the prepared chitosan/gelatin membranes are bioactive and are suitable for cell adhesion suggesting that these membranes can be used for tissue-engineering applications. Therefore, these novel chitosan/gelatin membranes are useful for biomedical applications.