Wentao Liu

Two-dimensional infrared spectroscopic study on the thermally induced structural changes of glutaraldehyde-crosslinked collagen.

The thermal stability of collagen solution (5 mg/mL) crosslinked by glutaraldehyde (GTA) [GTA/collagen (w/w)=0.5] was measured by differential scanning calorimetry and Fourier transform infrared spectroscopy (FTIR), and the thermally induced structural changes were analyzed using two-dimensional (2D) correlation spectra. The denaturation temperature (Td) and enthalpy change (ΔH) of crosslinked collagen were respectively about 27°C and 88 J/g higher than those of native collagen, illuminating the thermal stability increased. With the increase of temperature, the red-shift of absorption bands and the decreased AIII/A1455 value obtained from FTIR spectra indicated that hydrogen bonds were weakened and the unwinding of triple helix occurred for both native and crosslinked collagens; whereas the less changes in red-shifting and AIII/A1455 values for crosslinked collagen also confirmed the increase in thermal stability. Additionally, the 2D correlation analysis provided information about the thermally induced structural changes. In the 2D synchronous spectra, the intensities of auto-peaks at 1655 and 1555 cm(-1), respectively assigned to amide I band (CO stretching vibration) and amide II band (combination of NH bending and CN stretching vibrations) in helical conformation were weaker for crosslinked collagen than those for native collagen, indicating that the helical structure of crosslinked collagen was less sensitive to temperature. Moreover, the sequence of the band intensity variations showed that the band at 1555 cm(-1) moved backwards owing to the addition of GTA, demonstrating that the response of helical structure of crosslinked collagen to the increased temperature lagged. It was speculated that the stabilization of collagen by GTA was due to the reinforcement of triple helical structure.

Changes in aggregation behavior of collagen molecules in solution with varying concentrations of acetic acid

A critical aggregation concentration of 0.30-0.50mg/mL was previously obtained for type I collagen at 0.1M acetic acid (AA). In the present study, the aggregation behavior of collagen in solution (0.5mg/mL) in the presence of 0.1-2.0M AA was investigated. Circular dichroism showed that the three helix structure was maintained across the whole AA concentration range. However, the ratio of positive peak intensity over negative peak intensity varied depending on the conformational state of collagen aggregates. Ultra-sensitive differential scanning calorimetry revealed that transition temperatures Tm1 and Tm2 decreased by 8.35°C and 7.80°C, respectively, between 0.1M and 2.0M, indicating a possible relationship between the aggregation state and the thermal effect. The surrounding polarity of collagen molecules in solution containing pyrene was investigated by fluorescence spectroscopy, which demonstrated that disaggregation of collagen aggregates was enhanced with increasing AA concentration. This observation was correlated with changes in collagen fiber size observed by atomic force microscopy. Furthermore, collagen tyrosine residues were blue-shifted in an intrinsic fluorescence spectra, further indicating changes in aggregation behavior with increasing AA concentration. Finally, the dynamic response of collagen molecules to AA was analyzed by two-dimensional correlation fluorescence spectra.

The effect of glycerol and 2-propanol on the molecular aggregation of collagen in solution

The molecular aggregation of collagen solution (Col) in the presence of glycerol (Col-G) and 2-propanol (Col-P) was investigated. The transition temperature (Tm) of collagen, determined by ultra-sensitive differential scanning calorimetry, increased proportionally to the concentration of glycerol about 0.929 °C per 1M, while Tm decreased proportionally to the concentration of 2-propanol about 1.638 °C per 1M. The fluorescence spectra of collagen in solutions were detected by two-dimensional technique on the perturbation of the concentration of glycerol and 2-propanol and showed different results in Col-G and Col-P. The size of collagen aggregates in solutions was observed by dynamic light scattering. Compared with the aggregate size in Col, collagen aggregates became smaller in the presence of glycerol, whereas it grew larger in the presence of 2-propanol. Furthermore, the different extent of aggregation in Col, Col-G and Col-P influenced the self-assembly of collagen molecules under physiological conditions, and there were differences in the resulted fibril structures of Col, Col-G and Col-P after fibrillogenesis as determined by scanning electron microscopy. These results indicated that glycerol inhibited the collagen aggregates while 2-propanol promoted them in solution.

Structural properties of pepsin-solubilized collagen acylated by lauroyl chloride along with succinic anhydride

The structural properties of pepsin-solubilized calf skin collagen acylated by lauroyl chloride along with succinic anhydride were investigated in this paper. Compared with native collagen, acylated collagen retained the unique triple helix conformation, as determined by amino acid analysis, circular dichroism and X-ray diffraction. Meanwhile, the thermostability of acylated collagen using thermogravimetric measurements was enhanced as the residual weight increased by 5%. With the temperature increased from 25 to 115 °C, the secondary structure of native and acylated collagens using Fourier transform infrared spectroscopy measurements was destroyed since the intensity of the major amide bands decreased and the positions of the major amide bands shifted to lower wavenumber, respectively. Meanwhile, two-dimensional correlation spectroscopy revealed that the most sensitive bands for acylated and native collagens were amide I and II bands, respectively. Additionally, the corresponding order of the groups between native and acylated collagens was different and the correlation degree for acylated collagen was weaker than that of native collagen, suggesting that temperature played a small influence on the conformation of acylated collagen, which might be concluded that the hydrophobic interaction improved the thermostability of collagen.

Influence on the physicochemical properties of fish collagen gels using self-assembly and simultaneous cross-linking with the N-hydroxysuccinimide adipic acid derivative

Abstract Collagen gels from Southern catfish (Silurus meridionalis Chen) skins were prepared via the self-assembly of collagen molecules and simultaneous cross-linking with the N-hydroxysuccinimide adipic acid derivative (NHS-AA). The doses of NHS-AA were converted to [NHS-AA]/[NH2] ratios (0.025–1.6, calculated by the [active ester group] of NHS-AA and [ε-NH2] of lysine and hydroxylysine residues of collagen). When the ratio < 0.05, collagen gels were formed by collagen molecule self-assembly, resulting in the opalescent appearance of collagen gels and the characteristic D-periodicity of partial collagen fibrils, the collagen gel ([NHS-AA]/[NH2] = 0.05) displayed a small increase in denaturation temperature (Td, 42.8 °C), remaining weight (12.59%), specific water content (SWC 233.7) and elastic modulus (G′ 128.4 Pa) compared with uncross-linked collagen gel (39.1 °C, 9.12%, 222.4 and 85.4 Pa, respectively). As the ratio > 0.05, disappearance of D-periodicity and a gradual change in appearance from opalescent to transparent suggested that the inhibition of NHS-AA in the self-assembly of collagen molecules was more obvious. As a result, the collagen gel ([NHS-AA]/[NH2] = 0.2) had the lowest Td (35.8 °C), remaining weight (7.96%), SWC (130.9) and G′ (31.9 Pa). When the ratio was 1.6, the collagen molecule self-assembly was markedly suppressed and the formation of collagen gel was predominantly via the covalent cross-linking bonds which led to the transparent appearance, and the maximum values of Td (47.0 °C), remaining weight (45.92%) and G′ (420.7 Pa) of collagen gel. These results indicated that collagen gels with different properties can be prepared using different NHS-AA doses.

Rheological behavior of acylated pepsin-solubilized collagen solutions: Effects of concentration

Effects of concentration on the rheological behavior of acylated pepsin-solubilized collagen solutions were investigated by steady shear tests, dynamic frequency sweep, creep tests and thixotropic loop measurements in this paper. The results showed that both acylated collagen and native collagen solutions exhibited the typical pseudoplastic behavior and displayed shear thinned behavior with the increase of shear rate. With the increase of acylated collagen concentrations from 5 to 10 mg/mL, shear viscosity, elasticity modulus (G′), viscous modulus (G″), complex viscosity (η*), and the ability to resist deformation increased due to the physical entanglement, whilst loss tangent (tan δ) decreased. Additionally, with the increase of acylated collagen concentrations, the area of thixotropic loop increased from 6.94 to 44.40 watts/m3, indicating that the thixotropy of acylated collagen increased. Compared with native collagen solution, acylated collagen solution had stronger shear viscosity, η*, thixotropy, and ability to resist deformation. Furthermore, Power law model, Carreau model, Cross model, Leonov model and Burger model, were suitable for the fitting of the experimental data.

Investigation on the behavior of collagen self-assembly in vitro via adding sodium silicate

Silicon, a trace element found in human body, plays a critical role in the process of collagen self-assembly. In this study, the intermolecular interaction and fibrillogenesis process were investigated to understand the effects of various concentrations of sodium silicate (SS) on collagen self-assembly in vitro. Fourier transform infrared spectroscopy analysis indicated that the triple helical structure of collagen was not significantly affected by SS. Hydrophobic interactions and particle sizes of collagen aggregates, which were measured using pyrene fluorescence and dynamic light scanning, enhanced via adding 2 mM SS whereas decreased with further increasing concentrations (4-8 mM). Kinetic analysis revealed that an increase in hydrophobic interactions boosted collagen self-assembly in the presence of 2 mM SS. The inhibition of self-assembly with the addition of 4-8 mM SS, as illustrated by a reduction in the fibrillogenesis rate and turbidity, was potentially attributed to weak hydrophobic interactions and strong electrostatic repulsion. The observation of microscopy demonstrated that the fibrils exhibited the characteristic D-periodicity at 2 mM SS. The inhibitory effect of 4 mM SS was slight and the fibrils still formed, while the microstructure was consisted of clustered collagen aggregates as SS ≥ 6 mM owing to serious inhibition on collagen self-assembly.