Study on the structure and properties of wool keratin regenerated from formic acid

Structural characteristics of keratin regenerated from water (KW) and from formic (KF) acid solutions were compared. Amino acid composition and molecular weight distribution of KW and KF samples were studied by high performance liquid chromatography (HPLC) and SDS-PAGE electrophoresis. Turbidity measurement showed that keratin dissolved in formic acid forms transparent and stable solutions and no flocculation occurs. In addition, because of its good solvation properties, studied by viscosity measurements, formic acid can be used as a co-solvent to prepare keratin-based blend solutions. Structural studies carried out by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) and near infrared (NIR) suggest that formic acid stabilizes the beta-sheet structure. Thermogravimetric analysis (TGA) reveals a higher thermal stability of keratin regenerated from formic acid with respect to keratin regenerated from water.     

Study on cast membranes and electrospun nanofibers made from keratin/fibroin blends

Keratin regenerated from wool and fibroin regenerated from silk were mixed in different proportions using formic acid as the common solvent. Both solutions were cast to obtain films and electrospun to produce nanofibers. Scanning electron microscopy investigation showed that, for all electrospun blends (except for 100% keratin where bead defects are present), the fiber diameter of the mats ranged from 900 (pure fibroin) to 160 nm (pure keratin). FTIR and DSC analysis showed that the secondary structure of the proteins was influenced by the blend ratios and the process used (casting or electrospinning). Prevalence of beta-sheet supramolecular structures was observed in the films, while proteins assembled in alpha-helix/random coil structures were observed in nanofibers. Higher solution viscosity, thinner filaments, and differences in the thermal and structural properties were observed for the 50/50 blend because of the enhanced interactions between the proteins.     

Biodegradable materials based on silk fibroin and keratin

Wool and silk were dissolved and used for the preparation of blended films. Two systems are proposed: (1) blend films of silk fibroin and keratin aqueous solutions and (2) silk fibroin and keratin dissolved in formic acid. The FTIR spectra of pure films cast from aqueous solutions indicated that the keratin secondary structure mainly consists of alpha-helix and random coil conformations. The IR spectrum of pure SF is characteristic of films with prevalently amorphous structure (random coil conformation). Pure keratin film cast from formic acid shows an increase in the amount of beta-sheet and disordered keratin structures. The FTIR pattern of SF dissolved in formic acid is characteristic of films with prevalently beta-sheet conformations with beta-sheet crystallites embedded in an amorphous matrix. The thermal behavior of the blends confirmed the FTIR results. DSC curve of pure SF is typical of amorphous SF and the curve of pure keratin show the characteristic melting peak of alpha-helices for the aqueous system. These patterns are no longer observed in the films cast from formic acid due to the ability of formic acid to induce crystallization of SF and to increase the amount of beta-sheet structures on keratin. The nonlinear trend of the different parameters obtained from FTIR analysis and DSC curves of both SF/keratin systems indicate that when proteins are mixed they do not follow additives rules but are able to establish intermolecular interactions. Degradable polymeric biomaterials are preferred candidates for medical applications. It was investigated the degradation behavior of both SF/keratin systems by in vitro enzymatic incubation with trypsin. The SF/keratin films cast from water underwent a slower biological degradation than the films cast from formic acid. The weight loss obtained is a function of the amount of keratin in the blend. This study encourages the further investigation of the type of matrices presented here to be applied whether in scaffolds for tissue engineering or as controlled release drug delivery vehicles.     

Effect of formic acid exposure on keratin fiber derived from poultry feather biomass

Converting poultry feather biomass into useful products presents a new avenue of utilization of agricultural waste material. However, not much is understood about the poultry feather structure or methods to process it. In this study, formic acid vapor is systematically allowed to penetrate the feather fiber structure, which is composed of keratin. The diffusion kinetics show Fickian behavior during absorption. After very long times, i.e., greater than 10(3)h, the absorption experiments are stopped and the formic acid is allowed to desorb from the keratin material. The desorption kinetics of formic acid out of the keratin fiber do not mirror the absorption kinetics, indicating a change in the keratin microstructure. DSC and NMR spectroscopy analyses on the keratin fiber show a reduction in the area of the crystalline melting peak and solubilization of amino acids upon formic acid exposure. This indicates that the crystallinity is disrupted resulting in more amorphous fraction in the keratin polymer.     

Formic acid triggers the "Acid Crash" of acetone-butanol-ethanol fermentation by Clostridium acetobutylicum

Solvent production by Clostridium acetobutylicum collapses when cells are grown in pH-uncontrolled glucose medium, the so-called "acid crash" phenomenon. It is generally accepted that the fast accumulation of acetic acid and butyric acid triggers the acid crash. We found that addition of 1 mM formic acid into corn mash medium could trigger acid crash, suggesting that formic acid might be related to acid crash. When it was grown in pH-uncontrolled glucose medium or glucose-rich medium, C. acetobutylicum DSM 1731 containing the empty plasmid pIMP1 failed to produce solvents and was found to accumulate 0.5 to 1.24 mM formic acid intracellularly. In contrast, recombinant strain DSM 1731 with formate dehydrogenase activity did not accumulate formic acid intracellularly and could produce solvent as usual. We therefore conclude that the accumulation of formic acid, rather than acetic acid and butyric acid, is responsible for the acid crash of acetone-butanol-ethanol fermentation.     

Alpha-helix formation by solvent-solvent interaction

The degree of helicity θ of a series of homologous polypeptides as a function of solvent composition was investigated. The polypeptides studied were: poly-N⁵-(3-hydroxypropyl)-L -glutamine (PHPG) as well as the corresponding 2-hydroxyethyl and 4-hydoxybutyl derivatives (PHKG and PHBG, respectively). PHPG, which is nonhelical in formic acid, attains helicity on addition of relatively small amounts of formates, formamide, and urea to its solution in formic acid. This demonstrates that the high acidity of pure formic acid is largely responsible for its helix-breaking power-probably through protonation of the peptide bonds. In formic acid-water mixtures all three polymers show a maximum in degree of helicity at a mole fraction of about 0.3 formic acid. This is interpreted as being due to interaction between the two helix-breaking solvents, which results in the formation of an inactive molecular species. It is shown that solvent-induced transitions with helicity maxima are predicted by the Bixon-Lifson treatment when applied to this system.     

The role of formic acid in solution stability and crystallization of silk protein polymer

In this paper, the regenerated silk fibroin (SF) solution dissolved in formic acid was used as a model protein to understand the role of formic acid in solution stability and crystallization of protein-based materials. The molecular decomposition of SF did not occur for the dissolution process in formic acid within 1–2 days of storage times. The β-sheet crystallization of SF molecules was occurred by the elimination of formic acid upon drying. The SF molecules in formic acid solution are stable and have low hydrodynamic radius values. This may be closely related to the fact that formic acid has two opposite functions of dissolution and crystallization simultaneously. The turbidity, dynamic light scattering and FTIR measurements elucidate that the solution stability and crystallization of SF are attributed to compact molecular shape of SF in formic acid, resulted from the molecular interactions between formic acid and polar groups in SF molecules.     

Effects of acetic and formic acid on ABE production by <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> and <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

The effect of acetic acid and formic acid on acetone-butanol-ethanol (ABE) production by solventogenic Clostridia was investigated. The ABE concentration in Clostridium acetobutylicum was found to have increased slightly on addition of 3.7 ∼ 9.7 g/L acetic acid, but was found to have drastically reduced in the presence of 11.7 g/L acetic acid. However, the solvent production of C. beijerinckii was not affected by addition of acetic acid in the range of 3.7 ∼ 11.7 g/L. C. acetobutylicum was more vulnerable to formic acid than C. beijerinckii. In C. acetobutylicum, the total ABE production decreased to 77% on addition of 0.4 g/L formic acid and 25% with 1.0 g/L formic acid. The total ABE production by C. acetobutylicum was also noted to have decreased from 15.1 to 8.6 g/L when 8.7 g/L acetic acid and 0.4 g/L formic acid co-existed. The solvent production by C. beijerinckii was not affected at all under the tested concentration range of formic acid (0.0 ∼ 1.0 g/L) and co-presence of acetic acid and formic acid. Therefore, C. beijerinckii is more favorable than C. acetobutylicum when the ABE is produced using lignocellulosic hydrolysate containing acetic and formic acid.     

How to develop globular proteins into adhesives

To make globular proteins suitable for application in adhesives, the specific bonds and interactions which shape their structure have to broken. Only then, a layer of relatively large, flexible and interwoven polymer chains, which are firmly attached to the solid surface by adsorption, can be created. Such a network layer is essential to save the adhesive bond under an applied force, because it can distribute the concentration of stresses generated at the interface into the bulk. Unfolding and swelling of a protein can be achieved by changing the solvent quality. For the globular whey protein beta-lactoglobulin, the optimal conditions for unfolding and swelling is found with 98% formic acid as a solvent. In formic acid, beta-lactoglobulin looses its amphoteric character (it is protonated, probably for approximately 20%). In addition, formic acid is less polar than water and thus a better solvent for the apolar parts of the protein. The swelling and unfolding behaviour of beta-lactoglobulin is studied by viscosity and CD-spectroscopy measurements. For the interpretation of the results we apply the Kuhn formalism that the conformation of a protein can be described in terms of a statistical chain which consists of segments of an average persistence length P. The statistical segment length P, which varies with the experimental conditions, is directly related to the adsorption energy required for a strong adhesion between coil and surface. It determines the depletion energy kT P(-2) m(-2) which must be overcome by specific attraction between side groups of the protein chain and the surface. For beta-lactoglobulin in 98% formic acid, we find a P value of approximately 2.2 nm, pointing at a relatively flexible chain. The minimum net adsorption energy kT P(-2) is then approximately 1 mJ m(-2), a relatively small value to be exceeded. Preliminary results of destructive adhesion tests on beech wood lap-shear joints reveal promising tensile strengths of approximately 2.9+/-1.1 N mm(-2), indeed.     

Uracil in formic acid hydrolysates of deoxyribonucleic acid

1. When DNA is hydrolysed with formic acid for 30min. at 175 degrees and the hydrolysate is chromatographed on paper with propan-2-ol-2n-hydrochloric acid, in addition to expected ultraviolet-absorbing spots corresponding to guanine, adenine, cytosine and thymine, an ultraviolet-absorbing region with R(F) similar to that of uracil can be detected. Uracil was separated from this region and identified by its spectra in acid and alkali, and by its R(F) in several solvent systems. 2. Cytosine, deoxyribocytidine and deoxyribocytidylic acid similarly treated with formic acid all yielded uracil, as did a mixture of deoxyribonucleotides. 3. Approx. 4% of deoxyribonucleotide cytosine was converted into uracil by the formic acid treatment.     

Structural studies of Bombyx mori silk fibroin during regeneration from solutions and wet fiber spinning

Regenerated silk fibroin materials show properties dependent on the methods used to process them. The molecular structures of B. mori silk fibroin both in solution and in solid states were studied and compared using X-ray diffraction, FTIR, and (13)C NMR spectroscopy. Some portion of fibroin protein molecules dissolved in formic acid already have a beta-sheet structure, whereas those dissolved in TFA have some helical conformation. Moreover, fibroin molecules were spontaneously assembled into an ordered structure as the acidic solvents were removed from the fibroin-acidic solvent systems. This may be responsible for the improved physical properties of regenerated fibroin materials from acidic solvents. Regenerated fibroin materials have shown poor mechanical properties and brittleness compared to their original form. These problems were technically solved by improving the fiber forming process according to a method reported here. The regenerated fibroin fibers showed much better mechanical properties compared to the native silk fiber and their physical and chemical properties were characterized by X-ray diffraction, solid state (13)C NMR spectroscopy, SinTech tensile testing, and SEM.     

甲酸乙酯及甲酸对离体玉米象乙酰胆碱酯酶及酯酶活性的影响

研究甲酸乙酯和甲酸对离体玉米象Sitophilus zeamais（Motschulsky）酯酶和乙酰胆碱酯酶（AChE）的抑制作用,结果表明甲酸乙酯对离体的玉米象酯酶和AChE没有抑制作用,并且甲酸对玉米象酯酶和AChE的抑制中浓度远远高于甲酸乙酯对玉米象的致死中浓度,说明甲酸对玉米象这两种酶的抑制作用不是甲酸乙酯杀虫的主要机理。     

A differential dilatometer

To obviate the difficulties resulting from partial solubility of membrane proteins in detergents or from the use of noxious solvent mixtures containing phenol or chloral, a simple procedure was devised for acrylamide gel electrophoresis of membrane proteins in 13 m formic acid. Polyacrylamide gels are equilibrated in 13 m formic acid and used in the electrophoresis assembly with 13 m formic acid as the electrolyte. Particulate proteinaceous preparations are dissolved in trifluoroacetic acid or in 24 m formic acid containing glycine to increase the density and to facilitate the solubilization of the protein. Protein samples (10 to 100 μg) migrate as polycations.     

Proteomic analysis optimization: selective protein sample on-column retention in reverse-phase liquid chromatography

In an effort to optimize reverse-phase liquid chromatography (RPLC) for proteomics, we studied the impact of composition of the sample injection solution on protein on-column selection and retention. All the proteins studied were retained on-column when injections were made in 50% formic acid, 0.1% TFA or 8.3M urea. When formic acid was increased to 80%, the superoxide dismutase standard (MW 26,159) and 58 mouse microsomal proteins that possessed low-range molecular weights, high pIs or basic amino acid clusters were non-retained, resulting in retention selectivity during sample injection. Introducing to the 80% formic acid injection solution an organic solvent such as acetonitrile or acetonitrile-DMSO induced further retention selectivity, and increasing levels of organic solvents reduced on-column retention. The proteome was split into the proteins that were retained on-column which eluted at higher retention times (RTs), vs the proteins that collected in the injection flow-through which normally eluted at lower RTs. This protein selectivity was confirmed after fraction collection, 1D-GE and nano-LC-MS/MS. The significance of this procedure is that it can be exploited for fast extraction of small basic proteins from the bulk of the proteome and for on-column enrichment of hydrophobic proteins.     

Human red cell galactose 1-phosphate uridylyltransferase: effects of site-specific reagents on catalytic activity

Egg shell membrane protein was found to contain the crosslinking amino acids desmosine and isodesmosine. Of particular interest, the desmosine and isodesmosine content was increased severalfold when the egg shell membrane protein was subjected to autoclaving. The major protein in membranes, which contains the crosslinking amino acids desmosine and isodesmosine, differs greatly from elastin in amino acid composition and is resistant to digestion with elastase. It is concluded that this protein component is not elastin but contains desmosine isomers. Further, its amino acid composition does not resemble those reported for other fibrous proteins such as keratin, connectin, collagen, or microfibrillar protein.     

New defensive chemical data for ground beetles (Goleoptera: Carabidae): interpretations in a phylogenetic framework

First reports of major defensive chemicals for ground beetles representing four tribes include: Morionini (formic acid), Dercylini (methacrylic and tiglic acids), Catapieseini (formic acid and decyl acetate) and Perigonini (formic acid and dccyl acetate). Multiple species from Loxandrini were sampled and, shown to contain formic acid, not salicylaldehyde as previously reported. Several hexenoic acid compounds were found in the clivinine genus Schizogenius representing a third class of chemicals for that tribe. Salicylaldehyde was found for the first time in a species of Oodini. Additional species from Pterostichini, Patrobini and Odacanthini were sampled and the results were found to be consistent with previously published reports. The taxonomic distribution of defensive secretions is reviewed for tribes across the family Carabidae. The simultaneous occurrence of hydrocarbons and formic acid is noted in phylogenetically more derived carabids. By mapping chemical classes onto a phylogenetic hypothesis, it is shown that formic acid or other relatively strong irritants are correlated with tribes having a high species diversity in tropical regions, whereas tribes exhibiting higher diversity in temperate regions use milder saturated/unsaturatcd earboxylic acids. Based on this phylogenetic interpretation, the evolution and maintenance of formic acid is interpreted as the result of predation pressures and possibly the evolution of chemical mimicry.     

Use of weak acids to determine the bulk diffusion limitation of H+ ion conductance through the gramicidin channel.

The addition of 2 M formic acid at pH 3.75 increased the single channel H+ ion conductance of gramicidin channels 12-fold at 200 mV. Other weak acids (acetic, lactic, oxalic) produce a similar, but smaller increase. Formic acid (and other weak acids) also blocks the K+ conductance at pH 3.75, but not at pH 6.0 when the anion form predominates. This increased H+ conductance and K+ block can be explained by formic acid (HF) binding to the mouth of the gramicidin channel (Km = 1 M) and providing a source of H+ ions. A kinetic model is derived, based on the equilibrium binding of formic acid to the channel mouth, that quantitatively predicts the conductance for different mixtures of H+, K+, and formic acid. The binding of the neutral formic acid to the mouth of the gramicidin channel is directly supported by the observation that a neutral molecule with a similar structure, formamide (and malonamide and acrylamide), blocks the K+ conductance at pH 6.0. The H+ conductance in the presence of formic acid provides a lower bound for the intrinsic conductance of the gramicidin channel when there is no diffusion limitation at the channel mouth. The 12-fold increase in conductance produced by formic acid suggests that greater than 90% of the total resistance to H+ results from diffusion limitation in the bulk solution.     

Effect of Formic Acid on Conversion of Fructose to 5-Hydroxymethylfurfural in Aqueous/Butanol Media

Acid-catalyzed dehydration of carbohydrates into 5-hydroxymethylfurfural (HMF), a valuable biomass-derived intermediate, has received increasing attention. Efficient methods for HMF production are needed for successful commercialization of HMF in the near future. A new process for the dehydration of sugars into 5-hydroxymethylfurfural in aqueous/butanol media enhanced by using formic acid was developed. The effects of formic acid concentration, reaction temperature, and reaction time on the fructose conversion and HMF yield showed the significant influences of these process variables. The optimum conditions were found to be 2.5?mol/L formic acid concentration, 170°C and 70?min. Under such conditions, a fructose conversion of 98.3% with a HMF yield of 69.2% was achieved. The application of the butanol solvent and formic acid led to the conversion of fructose to HMF with high yield. The catalytic system in this study has prospects for commercial application due to its less corrosion and convenient downstream separation.     

Catabolic pathways of glucose in Bacillus circulans var. alkalophilus

Enzymes and the metabolic pathways of glucose catabolism of Bacillus circulans var. alkalophilus were studied. The metabolism of the microbe was mixed acid fermentative yielding mainly acetic and formic acids as end products from glucose. It was estimated that B. circulans var. alkalophilus partitions 90%–93% of the carbon from glucose into the Embden-Meyerhof-Parnas (EMP) pathway and 7%–10% into the hexose monophosphate (HMP) and Entner-Doudoroff (ED) pathways. Rather low activities of glucose dehydrogenase and gluconokinase appeared in the early logarithmic and late stationary phases, whereas NADH oxidase was markedly high. This result can be explained by a demand to reduce NADH to NAD⁺ for the EMP pathway; when acetic and formic acids are produced, no NADH is regenerated to NAD⁺, which is required in the early steps of EMP and HMP pathways. A small percentage (1.6%–2.4%) of the total CO₂ was formed from (6-C) of glucose, which means that the tricarboxylic acid cycle was functional but its contribution was insignificant. Large differences do not seem to exist between alkaliphilic and neutrophilic bacilli in the use of glucose pathways. Received: January 29, 1999 / Accepted: July 30, 1999