Eleanor M. Brown

Enzyme-catalyzed gel formation of gelatin and chitosan: potential for in situ applications.

We compared the ability of two enzymes to catalyze the formation of gels from solutions of gelatin and chitosan. A microbial transglutaminase, currently under investigation for food applications, was observed to catalyze the formation of strong and permanent gels from gelatin solutions. Chitosan was not required for transglutaminase-catalyzed gel formation, although gel formation was faster, and the resulting gels were stronger if reactions were performed in the presence of this polysaccharide. Consistent with transglutaminases ability to covalently crosslink proteins, we observed that the transglutaminase-catalyzed gelatin-chitosan gels lost the ability to undergo thermally reversible transitions (i.e. sol-gel transitions) characteristic of gelatin. Mushroom tyrosinase was also observed to catalyze gel formation for gelatin-chitosan blends. In contrast to transglutaminase, tyrosinase-catalyzed reactions did not lead to gel formation unless chitosan was present (i.e. chitosan is required for tyrosinase-catalyzed gel formation). Tyrosinase-catalyzed gelatin-chitosan gels were observed to be considerably weaker than transglutaminase-catalyzed gels. Tyrosinase-catalyzed gels were strengthened by cooling below gelatins gel-point, which suggests that gelatins ability to undergo a collagen-like coil-to-helix transition is unaffected by tyrosinase-catalyzed reactions. Further, tyrosinase-catalyzed gelatin-chitosan gels were transient as their strength (i.e. elastic modulus) peaked at about 5h after which the gels broke spontaneously over the course of 2 days. The strength of both transglutaminase-catalyzed and tyrosinase-catalyzed gels could be adjusted by altering the gelatin and chitosan compositions. Potential applications of these gels for in situ applications are discussed.

Processing of leather waste: pilot scale studies on chrome shavings. Isolation of potentially valuable protein products and chromium

Hides come to the tanner as a by-product of the meat industry. The tanning process, in turn, generates much greater quantities of by-products and wastes than leather. One ton of wet salted hides yields only 200 kg of leather but over 600 kg of solid waste, or by- product if a market can be found. In the United States, nearly 60,000 metric tons of chromium-containing solid waste, i.e. chrome shavings, are generated by the leather industry each year, and approximately ten times this amount is generated worldwide. Land application for the disposal of chromium-containing tannery and other leather wastes has been widely practiced during most of the twentieth century, but fewer landfill sites can be found every day and the cost of transportation and disposal increases. Historically, these materials were used in the production of fertilizer or composite boards, but while once the company producing and marketing fertilizer or boards would pay for the waste and its transportation, nowadays, the tanner has to pay for such things. Over several years, we have demonstrated that it is possible to isolate protein products (gelatin and collagen hydrolysate) from chrome shavings by using an alkaline protease under mild conditions. The objective of the present work was to perform pilot plant trials to isolate protein products from chrome shavings, treat and purify the remaining chrome cake and tan hides with the recovered chromium. Because of the high nitrogen content, the isolated collagen hydrolysate has potential use as a fertilizer and in animal feed additives. The gelatin has potential use in cosmetics, adhesives, printing, photography, microencapsulation, films or even as an additive in finishing products for the leather industry.

An energetic evaluation of a “Smith” collagen microfibril model

An energy minimized three-dimensional structure of a collagen microfibril template was constructed based on the five-stranded model of Smith (1968), using molecular modeling methods and Kollman force fields (Weiner and Kollman, 1981). For this model, individual molecules were constructed with three identical polypeptide chains ((Gly-Pro-Pro)n, (Gly-Prop-Hyp)n, or (Gly-Ala-Ala)n, wheren=4, 12, and 16) coiled into a right-handed triple-helical structure. The axial distance between adjacent amino acid residues is about 0.29 nm per polypeptide chain, and the pitch of each chain is approximately 3.3 residues. The microfibril model consists of five parallel triple helices packed so that a left-handed superhelical twist exists. The structural characteristics of the computed microfibril are consistent with those obtained for collagen by X-ray diffraction and electron microscopy. The energy minimized Smith microfibril model for (Gly-Pro-Pro)12 has an axial length of about 10.2 nm (for a 36 amino acid residue chain), which gives an estimated D-spacing (234 amino acids per chain) of approximately 66.2 nm. Studies of the microfibril models (Gly-Pro-Pro)12, (Gly-Pro-Hyp)12, and (Gly-Ala-Ala)12 show that nonbonded van der Waals interactions are important for microfibril formation, while electrostatic interactions contribute to the stability of the microfibril structure and determine the specificity by which collagen molecules pack within the microfibril.

Complex formation in sonicated mixtures of β-lactoglobulin and phosphatidylcholine

Abstractβ-Lactoglobulin, the major whey protein of bovine milk, is secreted via the endomembrane system of the mammary gland. The primary structure of β-lactoglobulin shares certain characteristics with membrane proteins, although the soluble protein assumes a globular conformation. We have prepared complexes of β-lactoglobulin and phosphatidylcholines by dissolving both in a helix-forming solvent (chloroform methanol). The complex is stable when transferred to aqueous solutions and sonicated to form vesicles. Both ionic and hydrophobic interactions appear to be involved in complex formation. We have used spectroscopy (circular dichroism, fluorescence, and nuclear magnetic resonance) and electron microscopy to study these complexes. At pH 3.7, the small, single bilayer vesicles produced by sonication are protected against aggregation by the presence of the protein. As determined by circular dichroism, the proportion of α-helix in β-lactoglobulin is increased by complexation with phosphatidylcholine. Circular dichroism and fluorescence spectra show the involvement of at least 1 tryptophan residue in the conformational change. At pH 7.2, β-lactoglobulin-phosphatidylcholine vesicles form aggregates as observed by electron microscopy and31P nuclear magnetic resonance spectroscopy. These aggregated vesicles could be resuspended by raising the pH. The ability of the partially unfolded β-lactoglobulin to interact with lipids is believed to be important to its transport through the endomembrane system.

‘New views’ on structure–function relationships in milk proteins

The molten globule state has been regarded as a major intermediate in protein folding. It is characterized by native-like secondary structure with a compact molecular size but little specific tertiary structure. α-lactalbumin under various denaturing conditions has been considered a paradigm of the classical molten globule state. It has been shown that caseins share many of the same properties and may therefore exist naturally in a molten globule-like state with defined secondary structure and limited fluctuating tertiary structure, which lead to their propensity for polymerization. The architectural concepts of tensegrity may be used to describe, in part, the structure of casein polymers.

Solution Structures of Casein Peptides: NMR, FTIR, CD, and Molecular Modeling Studies of αs1-Casein, 1–23

To determine its potential for interacting with other components of the casein micelle, the N-terminal section of bovine αs1-casein-B, residues 1-23, was investigated with nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies, and molecular modeling. NMR data were not consistent with conventional α-helical or β-sheet structures, but changes in N-H proton chemical shifts suggested thermostable structures. Both CD and FTIR predicted a range of secondary structures for the peptide (30–40% turns, 25–30% extended) that were highly stable from 5°C to 25°C. Other conformational elements, such as loops and polyproline II helix, were indicated by FTIR only. Molecular dynamics simulation of the peptide predicted 32% turns and 27% extended, in agreement with FTIR and CD predictions and consistent with NMR data. This information is interpreted in accord with recent spectroscopic evidence regarding the nature of unordered conformations, leading to a possible role of αs1-casein (1–23) in facilitating casein-casein interactions.

Influence of Neutral Salts on the Hydrothermal Stability of Acid-Soluble Collagen

The thermal stability of acid-soluble collagens was studied by circular dichroism (CD) spectroscopy. Adult bovine dermal collagen (BDC), rat-tail tendon collagen (RTC), and calf skin collagen (CSC) were compared. Despite some variability in amino acid composition and apparent molecular weight, the CD spectra for helical and unordered collagen structures were essentially the same for all the sources. The melting of these collagens occurs as a two-stage process characterized by a pretransition (Tp) followed by complete denaturation (Td). The characteristic temperatures vary with the source of the collagen; for mature collagens (BDC, RTC) Tp = 30°C and Td = 36deg;C, and for CSC Tp = 34°C and Td = 40°C. Neutral salts, NaCl or KCl, at low concentrations (0.02–0.2 M) appear to bind to the collagens and shift the thermal transitions of these collagens to lower temperatures.

Specificity of bovine heart protein kinase for the Δ-stereoisomer of the metal—ATP complex

We have shown, by kinetic and metal-binding studies that the catalytic subunit of bovine heart protein kinase tight& binds 2 divalent cations only in the presence of ~~c~eotjdes such as ATP [ I]_ The data were ~~er~re~ed to indicate that 1 of the 2 divafent cations interacts solely with the enzyme-bound ATP forming the active ternary enzyme-ATP-M” complex. As determined by NMlR, the other, somewhat less tightly-bound divalent cation, bridges the enzyme to the trip~~osph~t~ chain of the metal-ATP complex,. forming a h~~~y-i~ll~bjted

LACTOFERRIN CONFORMATION AND METAL BINDING PROPERTIES

Lactoferrin⌿ is an iron binding protein found in milk and other mammalian secretions. It has properties similar to the serum transferrins and to ovotransferrin of avian egg-white (reviewed by Feeney and Komatsu, 1966). These proteins specifically bind two moles of ferric ion per mole of protein. For each Fe(III) bound, one molecule of bicarbonate is incorporated into the complex (Masson and Heremans, 1968). All of these Fe(III) binding proteins have been found to have similar amino acid compositions, optical spectra and electron paramagnetic resonance spectra.

Keratin capped silver nanoparticles--synthesis and characterization of a nanomaterial with desirable handling properties.

We report for the first time the stabilization of silver nanoparticles in good yield, average diameter 3.5 nm, using wool keratin hydrolysates as stabilizers. The nanoparticles are extremely stable as a suspension and can be lyophilized into a powder and easily reconstituted in solvent with no change in spectral properties relative to the initial suspension. The nanoparticles interact with nitrogen and oxygen moieties of the keratin hydrolysates under the pH conditions used in the synthesis and appear to act as cross-linkers between adjacent chains. The product has excellent handling properties which we believe will make it a very attractive biocompatible coating/additive, providing prolonged antimicrobial efficacy to a wide variety of products such as textiles, plastics, paints, orthopedic devices and others.