Acetic Acid-soluble Collagen Research Articles

Extraction and characterization of chicken feet soluble collagen

Sodium chloride-soluble collagen (SSC), acetic acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from the skin of chicken feet and then characterized. PSC, ASC and SSC showed the yields of 49.10%, 14.49% and 1.13% (Based on lyophilized dry weight), respectively. PSC, ASC and SSC were characterized as type I collagen, containing α1 and α2 chains as well as β and γ-chains. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectra of PSC, ASC and SSC were similar, suggesting that they maintained their intact triple helical structure. PSC, ASC and SSC contained Gly as the major amino acid with high contents of Glu, Ala, Pro and Hyp. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of PSC, ASC and SSC revealed that their surface topography were similar. Dynamic elastic behavior in PSC, ASC and SSC was detected. PSC showed the largest elasticity. Temperature sweeps test indicated that PSC had the highest denaturation temperature, followed by ASC, and then by SSC. Proline hydroxylation of PSC was higher (45.8%) than that of ASC, and SSC and accordingly PSC showed the highest thermal stability. PSC showed the highest degree of hydrolysis compared to ASC and SSC.

Water holding capacity and collagen profile of bovine m. infraspinatus during postmortem ageing

Changes in the water holding capacity (WHC) and collagen profile in bovine m. infraspinatus during ageing in a vacuum at 3°C up to 20days were studied. Moisture, expressible water and total collagen content were determined in raw samples and muscles were then subjected to thermal treatment in steam. Moisture, collagen fractions (total, water-soluble, insoluble, acetic acid-soluble, total soluble) and cooking loss were determined in the cooked meat. Ageing time did not significantly influence WHC (expressible water content as a % of moisture), moisture content in raw and cooked meat or cooking loss. Between the 5th and 10th day of ageing a significant increase was noted in acetic acid-soluble collagen and total soluble collagen content along with a decrease in insoluble collagen content (p<0.05). A significant negative relationship was found between the expressible water content and the water-soluble collagen. It was concluded that connective tissue ageing of bovine m. infraspinatus might be finished after 10days.

Isolation and characterisation of collagen from elk antler velvet

Collagen was extracted from the antler velvet of elk (Cervus elaphus). Two types of collagen were prepared namely, acetic acid-soluble collagen and pepsin-soluble collagen. The electrophoretic patterns of both of the collagens showed that they were heterotrimeric, i.e. they consisted of α1α2α3. The total yield of the collagen obtained from the elk antler velvet was 12.1%. Amino acid analysis of the collagen by high-performance liquid chromatography showed that imino acid content such as that of proline and hydroxyproline was high, which might contribute to better visco-elastic properties. The peptide mapping of the collagens showed their similarity with porcine Type I collagen, thereby suggesting that the primary structure of both collagens is identical to that of porcine skin Type I collagen. The thermal denaturation temperature was 37°C, which is comparable to porcine Type I collagen and may also be as a result of high imino acid content.

In vitro fibrillogenesis of collagen type I in varying ionic and pH conditions

Collagen is the most abundant protein in the human body, and has primary roles in the formation of tendons, cartilage and bone, it provides mechanical strength to skin and indeed almost every organ and muscle is associated with a layer of collagen. It is thus a key component of the extracellular matrix. Here we have studied the in vitro fibrillogenesis of acetic acid-soluble collagen type I under physiological and varying non-physiological conditions by TEM from negatively stained specimens. At pH 2.5 the collagen heterotrimer remains soluble at increasing buffer concentrations and in the presence of increasing NaCl concentrations. At pH 4.5 molecular aggregates form at low NaCl concentrations, but at higher NaCl concentrations fibrils with a diffuse ~11nm banding are formed. At pH 7.0, initial molecular aggregates form at low NaCl concentrations that progressively form characteristic ~67nm D-banded collagen fibrils at intermediate NaCl concentrations that cluster to form thicker multi-fibril D-banded fibres in higher NaCl concentrations. By contrast, increasing concentrations of sodium phosphate at pH 7.0 leads to the formation of flexuous, unbanded fibrils at higher concentrations from the initial, loosely aggregated form of collagen. At higher pHs, the formation of D-banded fibrils is less efficient, particularly at pH 9.0. Thus at neutral pH, the presence of chloride anions, rather than sodium cations, is required for the production of D-banded collagen fibrils; higher than normal physiological chloride concentrations in the form of NaCl or Tris·HCl at neutral pH, but not phosphate buffer, can also lead to the efficient in vitro formation of D-banded collagen fibrils.

Influence of saline and pH on collagen type I fibrillogenesis in vitro: Fibril polymorphism and colloidal gold labelling

We have produced different collagen type I fibrils by in vitro fibrillogenesis of acetic acid-soluble collagen within the pH range 2.5–9.0, in the presence and absence of 150 mM NaCl. The varying relatively stable molecular assemblies and polymorphic fibrillar end-products produced after 24 h incubation have been assessed and compared by the TEM study of specimens negatively stained with uranyl acetate. In the presence of 150 mM NaCl, the assembly of collagen at low pH (2.5) leads to the formation of initial molecular aggregates that progressively link together at slightly higher pH (5.0) to form sub-fibrils and spindle-shaped D-banded bundles of sub-fibrils. At pH 6.0 these D-banded bundles aggregate into larger spindle-shaped fibrils with lateral misalignment of the D-banding across the bundle. However, at pH 7.0 and 8.0, in the presence of 150 mM NaCl, the characteristic parallel-sided mature D-banded collagen type I fibres are formed. At pH 9.0 more loosely formed parallel-sided D-banded collagen fibrils are present, within which the spindle-shaped sub-fibrils can be defined by negative staining more convincingly than at pH 7–8. In the presence of 50 mM buffer at pH 2.5, but absence of 150 mM NaCl, collagen type I forms disorganized periodic initial molecular aggregates, which have a tendency to link together to form sub-fibrils. Flexuous collagen type I sub-fibrils predominate at pH 5.0, alongside large spindle-shaped fibrils that possess a regular transverse ∼10 nm periodicity, with an oblique ∼67 nm periodicity, significantly different to the D-banding periodicity. At pH 7.0 and pH 8 in the absence of saline loosely-formed flexuous and spindle-shaped fibres co-exist, with underlying sub-fibrils visible, but at pH 9.0 only disorganized flexuous fibrillar aggregates are present. Colloidal gold labelling of the characteristic D-banded collagen type I fibrils with 5 nm and 2 nm chemically reactive gold particles reveals a periodic labelling pattern, which is not apparent with 10 nm and 15 nm gold particles, due to steric hindrance. The flexuous and spindle-shaped collagen fibrils also bind 2 nm gold particles in a specific manner. In all cases, the specific chemisorption of gold onto the collagen fibrils is probably determined by the availability of repeating amino acid side chains of the collagen molecules along the fibril surface. The controlled production of varying stable collagen type I fibrillogenesis products is likely to be of value within numerous areas of biotechnology, biology and medicine, including experimental biomineralization.

Effect of calcium ions on the solubility of muscular collagen and tenderness of beef meat.

The aim of the present study was to determine the effect of aqueous solutions of calcium chloride and calcium lactate on the solubility of beef collagen in raw and cooked meat. The content of total collagen, water- and acetic acid-soluble collagen and insoluble collagen was determined in beef samples. Shearing force was measured after heat treatment. It was found that calcium ions had no significant effect on the solubility of muscular collagen. An improvement in beef tenderness (27%-34%) caused by calcium ions did not result from breaking of the native structure of muscular collagen.

Possible Involvement of Aminotelopeptide in Self-assembly and Thermal Stability of Collagen I as Revealed by Its Removal with Proteases

The functions of aminotelopeptide and N-terminal cross-linking of collagen I were examined. Acetic acid-soluble collagen I (ASC) was purified from neonatal bovine skin and treated with three kinds of proteases. The amino acid sequencing analysis of the N terminus showed that ASC contained a full-length aminotelopeptide. Pepsin and papain cleaved the aminotelopeptide of the alpha1 chain at the same site and the aminotelopeptide of the alpha2 chain at different sites. Proctase-treated ASC lost the whole aminotelopeptide, and the N-terminal sequence began from the tenth residue inside the triple helical region. The rates of fibril formation of pepsin-treated ASC and proctase-treated ASC were the same and were slower than that of ASC. The denaturation temperatures, monitored by CD ellipticity at 221 nm, of ASC, pepsin-treated, or papain-treated collagens were the same at 41.8 degrees C. Proctase-treated ASC showed a lower denaturation temperature of 39.9 degrees C. We also observed the morphology of the collagen fibrils under an electron microscope. The ASC fibrils were straight and thin, whereas the fibrils of pepsin-treated ASC were slightly twisted, and the fibrils from papain- and proctase-treated ASC were highly twisted and thick. When the collagen gel strength was examined by a modified method of viscosity-measurement, ASC was the strongest, followed by pepsin-treated ASC, and papain- and proctase-treated ASCs were the weakest. These results suggest that the aminotelopeptide plays important roles in fibril formation and thermal stability. In addition, the functions of intermolecular cross-linking in aminotelopeptides may contribute to the formation of fibrils in the correct staggered pattern and to strengthening the collagen gel.

Correlation of tissue constituents with the acoustic properties of skin and wound

The purpose of this study was to compare measurements of ultrasound properties of skin and wound tissue with measurements of material properties such as total collagen concentration, acetic acid soluble collagen concentration, water concentration, and morphologic properties. Using a scanning laser acoustic microscope (SLAM), both ultrasonic speed and attenuation coefficient values were obtained for control skin (2–3 cm from the wound), for skin immediately adjacent to wounds (within 0.3 mm), as well as for wound tissue itself. The attenuation coefficient and speed measurements were lowest for wound tissue followed by adjacent skin and then control skin. As the wounds healed there appeared to be an increase in both speed and attenuation coefficient although the wound age at which these increases started and the length of time for which they continued varied from one dog to the next. The precision of duplicate sample measurement of wave speed was ±1.7% for control skin, whereas that for attenuation coefficient it was ±16%. Both ultrasonic speed and attenuation coefficient were directly correlated with tissue collagen concentration and inversely correlated with tissue water concentration ( p < 0.001). Attenuation coefficient correlated best ( r = 0.73) with acetic acid soluble collagen concentration which reflects the changes in collagen taking place during the repair process. These attenuation measurements made at 100 MHz using the SLAM were compared for control skin and wound samples with measurements made at 10–40 MHz using backscatter acoustic techniques (BAT). The tissue samples analyzed by each ultrasound technique were from adjacent locations on the animals.

Reconstitution of collagen fibrils in vitro; the assembly process depends on the initiating procedure

Electron microscopy and turbidimetry have been used to show that the reconstitution of native-type banded fibrils from a solution of acetic acid-soluble collagen (with intact telepeptides) can proceed by different assembly pathways, depending on the initiating procedure used to bring the collagen from a dissolved to a precipitating condition. Initiation requires raising the pH, temperature (T) and ionic strength (I) of the solution (here from pH 3.4, T=4° C, I=0.01 to pH 7.4, T=34° C, I=0.2). A widely used procedure is an initial increase in pH and I, followed by a rise in T (the ‘neutral start’ procedure); this leads, in the early stages, to the mesh of long thin non-banded filaments described by Gelman et al. 9. When, however, the initiation steps are performed in reverse order, first raising T and then increasing pH and I (the ‘warm start’ procedure), non-banded filaments are not found as an abundant long-lived intermediate in the assembly process: instead, native-type banded fibrils constitute most of the precipitated material from the earliest stages onwards, and fibril formation takes place more rapidly. The simultaneous raising of T, pH and I (in a third procedure) not only yields banded fibrils throughout the course of precipitation (with no accumulation of filaments) but also results in a further increase in the rate of fibril formation. Filament formation appears therefore to be triggered by exposure to cold neutral solvent during initiation. When exposure to cold neutral solvent is avoided, an alternative and more efficient mode of assembly, involving the simultaneous lateral and axial growth of D-periodic fibrils, occurs.

Skin lysyl oxidase activity is not rate limiting for collagen crosslinking in the glucocorticoid-treated rat.

Lysyl oxidase activity in the skin of rats receiving triamcinolone diacetate (12 mg/kg) for three consecutive days was decreased by sixty-four percent as compared to control values. A decrease of lysyl oxidase activity was observed twelve hours after the initial glucocorticoid injection. The decreased lysyl oxidase activity was accompanied by a forty-nine percent decrease of acetic acid extractable collagen. There was also a forty-two percent decrease in the alpha/beta ratio of the acetic acid soluble skin collagen of glucocorticoid-treated animals. These data indicate that although skin lysyl oxidase activity is decreased by glucocorticoid treatment, the crosslinking of acid extracted collagen as measured by the alpha/beta ratio and collagen solubility is increased. Accordingly lysyl oxidase activity is not rate limiting for collagen crosslink formation in the skins of rats treated with glucocorticoids.

Solubility of Spinal Ligament Collagen in Idiopathic and Secondary Scoliosis

Supraspinous and interspinous ligaments from patients with infantile and adolescent idiopathic scoliosis and from patients with secondary scoliosis were investigated for collagen content and cross-linking. No differences were demonstrated in the collagen content of ligaments from the idiopathic and secondary scoliosis groups. Collagen solubility studies were used to test for cross-linking defects. Similar amounts of neutral salt-soluble and acetic acid-soluble collagen were extracted from the ligaments of all diagnostic groups. Investigation of the normally insoluble collagen pool extracted from the ligaments of different diagnostic groups after "lyotropic relaxation" revealed no differences in the amount of collagen extracted or in its stability to alkaline denaturation. Control experiments using rat skin collagen showed that the lyotropic procedure solubilized undegraded alpha- and beta-polypeptide chains from the tissue. Reports from other laboratories have suggested the presence of a collagen defect in the skin of patients with adolescent idiopathic scoliosis. The results of the present investigation show that it is unlikely that a generalized defect of collagen cross-linking, or a specific defect of ligament collagen cross-linking, occurs in either infantile or adolescent idiopathic scoliosis.

Studies on Collagen of Bovine Cornea

Soluble collagen in bovine corneal stroma was determined by homogenizing and sequentially extracting the tissue with 0.45 m NaCl, pH 7.4, 0.5 m citrate buffer, pH 4.3, and 0.5 m acetic acid. Calf cornea contains about three times more soluble collagen than cow cornea. In both calf and cow, the salt-soluble fraction contains the most collagen. Though calf corneas contain citrate and acetic acid-soluble collagen fractions, they are reduced greatly in the cow cornea. Calf corneas were incubated with 3,4–3H-proline for estimation of collagen biosynthesis and the data show that cornea has the capacity for in vitro synthesis of collagen. The tissue utilized radioactive proline slightly better when stroma alone without the epithelium was used. The specific activity of collagen was high in the salt-soluble fraction and also in the dialyzed incubation medium (containing collagen and soluble protein leached from the corneas). With the in vitro system, radioactivity was detected in the insoluble fibrous collagen in the corneas. Chelating agents, antibiotics, and other metabolic inhibitors markedly inhibited biosynthesis of collagen and other proteins. The iron chelator, α, α’-dipyridyl, selectively inhibits biosynthesis of corneal collagen.

Connective Tissue XV. Amino Acid Composition of Soluble Collagen of Pig Uterus

Summary1. 1 M NaCl-soluble and 0.5 M acetic acid-soluble collagen were purified from pig uterus. The total yield was approximately 0.03% of the weight of the wet tissue. 2. After heat denaturation, the α- and β- components were separated on Sephadex G-200 and CM-cellulose column. Like the finding in human uterine collagen, less α- and more β- components were found in the salt soluble collagen of pig uterus than in the skin of the rat and human being. However, more α- and less β-component were found in the uterine collagen of pig than in that from the human being. 3. Pig uterine collagen contained a somewhat higher level of hydroxyproline and hydroxylysine than dermal collagen of rat and human being.

The denaturation of acetic acid-soluble calf-skin collagen. Changes in optical rotation, viscosity and susceptibility towards enzymes during serial denaturation in solutions of urea.

The denaturation of acetic acid-soluble calf-skin collagen. Changes in optical rotation, viscosity and susceptibility towards enzymes during serial denaturation in solutions of urea.

The presence of non-protein nitrogen in acetic acid-soluble calf-skin collagen.

The presence of non-protein nitrogen in acetic acid-soluble calf-skin collagen.

The origin of N-terminal residues in acetic acid-soluble calf-skin collagen.

Research Article| May 01 1962 The origin of N-terminal residues in acetic acid-soluble calf-skin collagen FS STEVEN; FS STEVEN Search for other works by this author on: This Site PubMed Google Scholar GR TRISTRAM GR TRISTRAM Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1962) 83 (2): 245–248. https://doi.org/10.1042/bj0830245 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation FS STEVEN, GR TRISTRAM; The origin of N-terminal residues in acetic acid-soluble calf-skin collagen. Biochem J 1 May 1962; 83 (2): 245–248. doi: https://doi.org/10.1042/bj0830245 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1962 The Biochemical Society1962 Article PDF first page preview Close Modal You do not currently have access to this content.

Acetic acid-soluble collagen in human scars

Acetic acid-soluble collagen in human scars

Effect of growth hormone on acetic acid-extractable collagen of hamster skin.

SummaryConcentration of acetic acid-extractable collagen in the skin of hamsters was used as a measure of the effect of growth hormone on collagen metabolism. The intraperitoneal administration of growth hormone increased the concentration of the acetic acid-soluble collagen in the skin of old hamsters.