Skip to main content
Collagen and Leather
Download PDF

The impact of N-terminal modification of PAA with different chain lengths on the structure, thermal stability and pH sensitivity of succinylated collagen

Collagen and Leather volume 6, Article number: 6 (2024) Cite this article

Abstract

The limitations of native collagen, such as thermal stability and solubility in physiological environments, can be improved by applying bioconjugation and synthetic chemistry techniques. However, the exquisite control of the modification site of collagen remains a challenge. In this work, pH-responsive poly(acrylic acid) (PAA) with different chain lengths was attached to the N-terminal α-amino groups of succinylated collagen using a site-specific modification strategy. Additionally, the structure, thermal stability, and pH sensitivity of succinylated collagen were explored. The modification rate of amino groups in the succinylated collagen-PAA bioconjugate (SPSC-PAA) was evaluated by the 2,4,6-trinitrobenzene sulfonic acid assay. The impact of N-terminal modification of PAA and its chain length on the thermal stability of collagen was explored by CD and DSC. These techniques revealed that the thermal stability of SPSC-Col is pH-responsive and closely related to the chain length of grafted PAA. The pH sensitivity of SPSC-PAA was further explored by rheology and turbidity. Subsquently, the critical pH and isoelectric point of SPSC-PAAs were also examined by turbidity and isoelectric point titration, respectively. This work provides a new insight into the N-terminal modification of collagen on its properties.

Graphical abstract

1 Introduction

Collagen is the most abundant structural protein in the skins of animals, accounting for 80–85% of dermal protein [1]. In addition, collagen is widely distributed in many tissues, such as tendons, blood vessels, cartilage, and the bones of vertebrates [2]. The characteristic triple helical structure of collagen is formed by two α1(I) polypeptide chains and one α2(I) polypeptide chain with a polyproline II-type conformation. The amino acid sequence of each polypeptide chain contains the repeating unit of Gly-X-Y, in which proline and hydroxyproline are always in the X and Y positions, respectively [3]. As a heterotrimer, type I collagen contains 96% triple helical domain, and the non-helical domains are located at both ends of the collagen, namely telopeptides (Fig. 1) [4]. Notably, pepsin can remove the telopeptides of collagen without destroying its triple helical structure. For example, tropocollagen (with terminal telopeptides) and atelocollagen (without terminal telopeptides) can be obtained from animal tissue via acid solubilization or enzyme-assisted acid solubilization methods [5, 6].

Fig. 1
figure 1

A schematic diagram showing the synthesis of SPSC-PAA and its pH-responsive behavior

Full size image

The triple helical structure of collagen is stabilized by hydrogen bonds, hydrophobic bonding, electrostatic attractions, and van der Waals forces among α-polypeptide chains [7, 8]. The triple helical structure is vital to the excellent characteristics of collagen, such as bioactivity and self-assembly properties. Under high-temperature conditions, the collagen denatures, as the intermolecular interaction between α-polypeptide chains is destroyed resulting in the dissociation of the triple helical structure. The denatured collagen products (such as collagen hydrolysate and gelatin) possess different properties such as loss of self-assembly ability, absence of conformation translation, smaller molecular sizes, and reduced ability to promote cell adhesion and proliferation [9, 10]. Therefore, enhancing the thermal stability of collagen is a concern in practical applications and is attracting great attentions. On the other hand, as a polyampholyte, the isoelectric point of collagen near a neutral pH value results in its poor solubility in physiological environments. This limits its application in the field of biomedical materials [11]. In short, the low thermal stability and poor solubility in physiological environments always limit the application of collagen, despite its biocompatibility, low immunogenicity, and biodegradability. With the development of synthetic chemistry and bioconjugation techniques, the functional groups of collagen can be modified by reactive moieties to improve the inherent performance of collagen and acquire novel functions [12]. For example, the solubility of collagen in physiological environments can be improved by succinylation of free amino groups [13,14,15]. Modification with temperature-responsive polymers can improve the denaturation temperature of collagen [16]. Although collagen-polymer conjugates have been extensively prepared and studied, attaching polymers to the precise site of collagen is still very difficult. Recently, the attachment of poly(N-isopropylacrylamide) (PNIPAAm) to the N-terminal of succinylated collagen was achieved successfully [12], suggestting that N-terminal modification of collagen can improve its thermal stability.

As a pH-responsive weak polyelectrolyte, poly(acrylic acid) (PAA) has been widely used for the preparation of biomaterials. For example, PAA has been used to regulate the mineralization of collagen [17]. It is worth noting that the collagen/PAA composites are typically prepared by simple blending or cross-linking methods. However, the attachment of PAA on the N-terminal of collagen has not yet been reported. The stretched conformation of PAA at high pH changes to a coiled conformation at low pH. This is due to the formation of intermolecular hydrogen bonds between H2O and PAA and the formation of intramolecular hydrogen bonds in PAA. The critical pH of PAA is about 4.7, at which point the conformation of PAA starts to change [18]. Modified collagen with pH-responsive PAA might alter its thermal stability at different pH conditions, owing to the conformation exchange of PAA. On the other hand, the number of carboxyl groups in SPSC was further improved by the modification of PAA. This enhancement would be beneficial for the solubility of collagen in physiological environments.

Here, the succinylated collagen-PAA bioconjugates were prepared to reveal the impact of PAA modification and the polymer length on its thermal stability, solubility and pH sensitivity. The side chain amino groups in collagen were modified with succinic anhydride. Subsequently, the N-terminal α-amino groups were further modified with polyacrylic acid (PAA) via a “grafting from” strategy based on the ATRP polymerization method. The modification process would be monitored with a 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay. The impact of PAA modification on the triple helix in collagen was studied by circular dichroism (CD). The pH sensitivity of succinylated collagen-PAA bioconjugate (SPSC-PAA) was explored by CD and rheology methods. The critical pH and isoelectric point of SPSC-PAA with different chain lengths of PAA were estimanted by turbidity and isoelectric point titration, respectively. The pH responsive SPSC-PAA might be applied to fabricate in situ gel systems.

2 Materials and methods

2.1 Materials

Similar to a previous report, tropocollagen (with terminal telopeptides, named ASC) and atelocollagen (without terminal telopeptides, named PSC) were prepared from grass carps (Ctenopharyngodon idellus) skins. These were prepared by the acid solubilization method and enzymes/acid solubilization method, respectively [19].

Succinic anhydride, acrylic acid (AA), β-alanine, 2,4,6-trinitrobenzenesulfonic acid, 2-Bromo-2-methylpropionyl bromide, N,N′-diisopropylcarbodiimide, 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), CuBr, N-hydroxysuccinimide were obtianed from Sigma-Aldrich (USA). All analytical grade chemicals and solvents were used directly without further purification. All solutions were prepared with MilliQ water (18.2 MΩ·cm).

2.2 Synthesis of SPSC

Initially, a 2.0 mg/mL ASC solution was prepared with acetic acid (0.5 mol/L). It was then dialyzed with PBS (pH 8). A small amount of dimethyl sulfoxide was used to dissolve succinic anhydride. This was transferred into the PBS solution of ASC and stirred at 4 °C. After the reaction, excess reagents were removed by dialysis with PBS to yield the succinylated ASC (SASC). The SASC solution was combined with 2% pepsin (800–2,500 unit/mg, EC 3.4.23.1, Sigma, USA) and stirred for 24 h to cleave telopeptides. Finally, PBS (pH 8) was used for dialysis of the reaction solution to produce the SPSC solution.

2.3 Synthesis of SPSC-PAA

SPSC-PAA was synthesized following a previous method [12]. Excess initiators (N-2-bromo-2-methylpropionyl-β-alanine N′-oxysuccinimide ester) were added to the SPSC solution. The N-terminal α-amino groups in collagen were modified after stirring for 24 h at 10 °C. Then, dialysis against PBS (pH 8) was done to purify the obtained SPSC-ini solution.

The SPSC-ini solution was degassed by bubbling with nitrogen for 30 min. Next, acrylic acid (AA)/initiator, HMTETA, and CuBr were added (ratio for SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) were 90 mol/1 mol/5 mol/5 mol, 230 mol/1 mol/5 mol/5 mol/, and 460 mol/1 mol/5 mol/5 mol, respectively). The solution was stirred at 10 °C for 24 h under nitrogen. Finally, the reaction mixture was purified by dialysis with water and lyophilized for storage in a refrigerator.

2.4 GPC assay

p-Toluenesulfonic acid (4.5 mmol/mL) solution was used to dissolve SPSC-PAA (20 mg). It was hydrolyzed for 72 h at 80 °C. Then, the obtained PAA was placed into a 1 kDa cutoff dialysis bag and dialyzed with water. Its molecular weight was determined by gel permeation chromatography (GPC) at a flow rate of 1 mL/min at 50 °C [20]. The analysis was conducted using the Agilent 1260 Series HPLC system with a refractive index detector. Polyethylene oxide, styrene, and 50 mmol/L LiBr solution in DMF served as the calibration standard, flow marker, and eluent, respectively. The numerical average molecular weight (Mn) and polydispersity index (PDI) of PAA were determined.

2.5 TNBS assay

The modification rate of modified collagen was estimated as described previously with some modifications [21]. Briefly, a 0.5 mL 0.5 wt% TNBS solution was added to a 1.0 mL 2.0 mg/mL sample solution in PBS. It was then stirred at 40 °C for 4 h. Subsequently, 1.5 mL 6 mol/L HCl was mixed with the resulting solution. This was incubated at 90 °C until it became clear. Diethyl ether was used to extract the reaction solution to remove excess reagent. Then, the UV absorbance of the reaction solution was measured with a UV-2450 spectrophotometer (Shimadzu, Japan). The control group was established by adding TNBS after the addition of HCl. The modification rate of each sample was calculated using the following formula: [Absorbance of native collagen at 346 nm − Absorbance of modified collagen at 346 nm]/Absorbance of native collagen at 346 nm × 100.

2.6 Circular dichroism (CD)

A 0.1 mg/mL sample solution was prepared by dissolving the sample sponge in acetic acid (0.5 mol/L). It was equilibrated at room temperature for 1 h. Then, the solution was placed into a quartz cuvette with a path length of 1 mm. The triple helix of the sample was examined by a J-800C CD spectrometer (JACSO, Hachioji, Japan) at a scan rate of 1 nm/s. Furthermore, the incubation temperature of the sample solution was increased from 20 to 60 °C at a rate of 1 °C/min. The thermal denaturation process of the sample was evaluated by monitoring the CD peak value change at 220 nm.

2.7 DSC

The lyophilized sample was swelled with 0.5 mol/L acetic acid. It was then transferred into an aluminum cell, and an empty cell was used as a reference. The thermal denaturation endothermic process of the sample was detected by differential scanning calorimetry (DSC-Q10, TA Instruments, New Castle, Delaware, USA) from 30 to 60 °C at a rate of 2 °C/min. The DSC thermogram was analyzed with the Universal Analysis 2000 4.5A software (TA Instruments/Waters LLC) to determine the melting temperature (Tm) of the sample.

2.8 Critical pH of SPSC-PAA

The pH of a 2.0 mg/mL SPSC-PAA solution in PBS was adjusted from 3 to 12 using 0.1 mmol/mL of HCl and NaOH. The transmittance of the system was measured at 310 nm. The derivative of the transmittance-pH curve of the sample solution was calculated to determine the critical pH as described in a previous report with some modifications [22].

2.9 pH sensitivity (Rheology)

A 1.0 mL ASC or SPSC-PAA solution (3.0 mg/mL, pH 4 or 8) was loaded between parallel plates (diameter is 40.0 mm) with a 1.0 mm gap. The edges were sealed with silicone oil to prevent moisture loss. An oscillation time sweep was conducted using a stress-controlled rheometer (AR2000, TA Instruments, New Castle, DE, USA) at 1% strain amplitude.

2.10 Isoelectric point

The sample was dissolved in PBS (pH 7.4) to yield a 0.1 mg/mL solution. It was then titrated with 0.1 mol/L HCl and 0.1 mol/L NaOH. The zeta potential of the sample was recorded in the pH range of 3–10 (a pH value interval of 0.5) using a zeta potential titration apparatus (Malven Zetasizer Nano ZS, UK). When the zeta potential of the sample solution reached 0, the corresponding pH was defined as its isoelectric point.

3 Results and discussion

3.1 Modification rate of SPSC-PAA

The succinylated collagen-PAA conjugate (SPSC-PAA) was produced using the site-specific N-terminal α-amino modification strategy (Fig. 1). The modification rate of amino groups in the samples was measured by the 2,4,6-trinitrobenzene sulfonic acid (TNBS) colorimetric assay. Free amino groups can react with TNBS to form the chromogenic TNP derivative. This reaction can be monitored by UV–Vis, with the maximum absorbance peak typically at about 346 nm. As previously reported, a lower maximum absorbance at 346 nm indicates fewer free amino groups, suggesting a higher modification rate in collagen [21]. As shown in Fig. 2A, tropocollagen with terminal telopeptides (ASC) showed the highest absorbance peak. The absorbance peak at 346 nm of atelocollagen without terminal telopeptides (PSC) was lower than that of ASC. This was due to the decrease in the amount of free amino groups, resulting from the removal of telopeptides. All amino groups in ASC were protected with excess succinic anhydride to produce succinylated ASC (SASC). This resulted in a much lower maximum absorbance at 346 nm, due to the succinylation of free amino groups in ASC [15]. The modification rate of SASC was 97% ± 1%. Subsequently, pepsin digested the telopeptides of SASC to produce succinylated PSC (SPSC). This process exposed new α-amino groups. Compared to SASC, the maximum absorbance of SPSC at 346 nm increased slightly. The modification rate of amino groups in SPSC was 91% ± 2%. This increase can be attributed to the newly α-amino groups formed by the removal of telopeptides. The newly formed α-amino groups were then modified with an ATRP initiator to produce the ATRP initiator-modified SPSC (SPSC-ini). Its maximum absorbance at 346 nm decreased again, and the modification rate was 97% ± 2%. These results indicate that the proportion of N-terminal α-amino groups was about 6% of the total amino groups. All of these were modified with ATRP initiators. Finally, in situ atom transfer radical polymerization of AA on the initiators of SPSC-ini was performed to produce SPSC-PAA. The modification rate of SPSC-PAA was found to be similar to that of SPSC-ini.

Fig. 2
figure 2

A The maximum absorbance peak at 346 nm of collagen and modified collagen in UV–Vis spectra based on TNBS colorimetric assay. B CD spectra of collagen and modified collagen based on the wavelength scanning mode

Full size image

3.2 Triple helical structure of SPSC-PAA

The triple helical structure is crucial distinctive characteristic of collagen, such as its bioactivity and self-assembly properties. It is essential for the applications of collagen. For example, products derived from denatured collagen (such as collagen hydrolysate and gelatin) exhibit weaker proliferation ability for cell adhesion/proliferation. They also show lower resistance to enzymatic degradation, compared to collagen [23]. Therefore, CD was used to study the impact of modification on the triple helical structure of collagen. As shown in Fig. 2B, native collagen (ASC and PSC) demonstrated a negative absorption peak and a positive absorption peak at 197 and 220 nm, respectively. These peaks were caused by the π-π* amide transition and the n–π* transition [11]. Notably, SACS, SPSC, SPSC-ini, and SPSC-PAA(m) (m indicating the medium chain length of PAA) also displayed similar CD curves to those of native collagen. This suggests that the triple helical structure of collagen was compromised by the modification of amino groups.

3.3 Thermal stability of SPSC-PAA

The thermal stability of collagen is a concern for its application, as the denatured products of collagen often lose their intrinsic molecular behavior and biological properties. Therefore, understanding the impact of N-terminal modification on the thermal stability of collagen is helpful. Here, the impact of PAA modification on the thermal stability of collagen was explored by CD. Figure 3A shows the variation trend of the CD value at 220 nm with the increasing temperature. All modified samples (SASC, SPSC, SPSC-ini, and SPSC-PAA(m)) showed S-type curves like native collagen (ASC and PSC). These curves can be divided into three stages. (1) the high plateau stage, indicating the stable phase of the triple helical structure of collagen; (2) the rapid decline stage, indicating the transformation phase from the triple helix to random polypeptide chains; (3) the low plateau stage, indicating the complete destruction of the triple helix. Based on differential CD analysis, the denaturation temperatures of ASC, PSC, SASC, SPSC, SPSC-ini, and SPSC-PAA(m) were determined as 42.3 ± 0.5 °C, 42.2 ± 0.4 °C, 35.1 ± 0.4 °C, 31.6 ± 0.5 °C, 30.2 ± 0.3 °C, and 31.4 ± 0.4 °C, respectively (Fig. 3B).

Fig. 3
figure 3

A The thermal denaturation curves of collagen and modified collagen based on CD peak values at 220 nm. B The derivative thermal denaturation curves of collagen and modified collagen. C DSC thermograms of collagen and modified collagen

Full size image

Then, the thermal stability of these samples was further tested by DSC. The melting temperature (Tm), attributed to the dissociation of the triple helical structure in collagen, could be observed in the DSC thermograms. As shown in Fig. 3C, the Tm of ASC, PSC, SASC, SPSC, SPSC-ini, and SPSC-PAA(m) was 45.9 °C, 41.9 °C, 37.9 °C, 36.5 °C, 34.7 °C, and 40.7 °C, respectively. These results align with the CD analysis, although some differences exist due to the analysis instruments. The denaturation temperature of collagen decreased after modification, which is consistent with previous reports [12, 16, 24].

It is well known that the triple helical structure is stabilized by hydrogen bonding, hydrophobic bonding, electrostatic attractions, and van der Waals forces among α-peptide chains [7, 8]. The amino groups in collagen play a crucial role in maintaining its triple helix [15]. Therefore, modifying these amino groups increases steric hindrance and disrupts the hydrogen bonding among the peptide chains of collagen, resulting in a decrease in thermal stability. According to the report of Mils et al., there is a thermally labile domain in the triple helical structure of collagen, which is critical for its denaturation process [25]. The denaturation process begins with the dissociation of this thermally unstable region. Subsequently, the unzipping of the triple helical structure occurs along the collagen axis. In our previous work, the denaturation temperature of SPSC could be increased by about 6 °C through PNIPAAm modification on its N-terminal α-amino groups [12]. However, in this work, we found that the modification of SPSC’s N-terminal α-amino groups with PAA did not significantly improve its thermal stability (denaturation temperatures of SPSC and SPSC-PAA(m) are 31.6 ± 0.5 °C and 31.4 ± 0.4 °C, respectively).

Therefore, we explored the causes of this phenomenon. Notably, PAA is a pH-responsive biocompatible polymer that is widely used in the fabrication of pH-responsive materials in biomaterials and drug delivery systems [26]. The carboxylic groups of PAA can accept and release protons at low and high pH, respectively. This results in a transformation between hydrophobic and hydrophilic states based on the balance between electrostatic repulsion forces and hydrogen bonding at different pH [27]. The thermal stability testing of SPSC-PAA was conducted at pH 8. In this state, the PAA grafted onto SPSC-PAA always remains in an extended hydrophilic state and cannot aggregate with temperature change. Thus, PAA could not enhance the stability of the collagen triple helical structure at high pH conditions.

To confirm this hypothesis, we studied the impact of PAA modification at different pH conditions (pH 8, 6, and 4) on the thermal stability of collagen. As shown in Fig. 4A, when pH changed from 8 to 6 and 4, the CD value-temperature curves of SPSC-PAA(m) were also S-type with high plateau, rapid decline, and low plateau stages. Furthermore, the differential CD analysis (Fig. 4B) revealed that the denaturation temperature of SPSC-PAA(m) at pH 6 was 31.6 ± 0.6 °C, similar to that at pH 8 (31.4 ± 0.4 °C). However, at pH 4, the denaturation temperature increased to 35.2 ± 0.5 °C. This was attributed to the aggregation of PAA at low pH. These results confirm that the aggregation of grafted polymers on N-terminal collagen crucial for the thermal stability of collagen.

Fig. 4
figure 4

A The thermal denaturation curves of SPSC-PAA(m) based on the CD peak values at 220 nm at pH 4, 6, and 8. B The derivative thermal denaturation curves of SPSC-PAA(m) at pH 4, 6, and 8

Full size image

3.4 pH sensitivities of SPSC-PAA

The mechanical properties of SPSC-PAA(m) and ASC were measured by rheology to evaluate the pH sensitivity of SPSC-PAA(m). The experiments were conducted at pH 4 and 8, which are lower and higher than the critical pH of SPSC-PAA(m), respectively. As shown in Fig. 5A, the elastic modulus (G′) of the ASC system was dominant in the range of testing frequencies, however, the G′ was less than 2 Pa, suggesting that the ASC system formed a weak elastic hydrogel. It is noteworthy that no obvious difference was observed in the mechanical properties of the ASC system when the pH changed from 8 to 4. As shown in Fig. 5B, at pH 8, the G′ of the SPSC-PAA(m) system was lower than the viscous modulus (G″), indicating a solvent-like behavior. However, the G′ of the SPSC-PAA(m) system increased to about 37 Pa at pH 4, which was higher than G″. The conformation of PAA changes from stretched at high pH to coiled at low pH. This is due to the shift form intermolecular hydrogen bonding between H2O and PAA to intramolecular hydrogen bonding in PAA [18]. Consquently, the SPSC system can convert between solution and gel states under different pH conditions. These results indicate the SPSC-PAA(m) system possesses pH sensitivity and can form an elastic hydrogel at pH 4. This makes it suitable for fabricating in situ gel systems. Additionally, the pH sensitivity of SPSC-PAA(m) confirms the successful modification of PAA on the N-terminal amino groups in collagen.

Fig. 5
figure 5

Elastic modulus (G′) and viscous modulus (G″) of rheological spectra of ASC (A) and SPSC-PAA(m) (B) at pH 4 and 8, respectively

Full size image

3.5 The impact of grafted polymer length on the triple helical structure of SPSC-PAA

It is well known that the properties of matter are closely related to their structure. Thus, the effect of grafted PAA chain length on the triple helical structure of SPSC-PAA was investigated. The PAA chain length of SPSC-PAA was controlled by adjusting the molar ratio of initiators and acrylic acids. The ratios were 1/90, 1/230, and 1/460 for SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l), respectively. Subsequently, the attached PAA chains were cleaved from SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l). This was characterized by GPC. The results indicated that the number average molecular weight (Mn) of PAA in SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) was 2379, 3893, and 5268 Da, with a PDI of 1.37, 1.12, and 1.18, respectively. Next, the integrity of the triple helical structure of SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) was characterized by CD. SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) displayed similar CD curves with negative and positive absorption peaks at 197 and 220 nm, respectively (Fig. 6). These finding suggested that the polymer chain length of PAA did not significantly affect on the integrity of the triple helix in collagen.

Fig. 6
figure 6

CD spectra of collagen, SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) based on the wavelength scanning mode

Full size image

Additionally, the impact of PAA chain length on the thermal stability of collagen at pH 4 was examined. In Fig. 7A, the variation trend in the CD value at 220 nm of SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) with increasing temperature was observed. The CD value-temperature curves of SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) were S-shaped with high plateau, rapid decline, and low plateau stages. In Fig. 7B, differential CD analysis revealed that the denaturation temperature of SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) at pH 4 was 33.2, 35.2, and 41.5 °C, respectively. Notably, the denaturation temperature of SPSC-PAA(l) was higher than that of ASC (38.1 °C) and PSC (37.4). The thermal stability of SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) was then assessed by DSC. As shown in Fig. 7C, the Tm of SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) was 36.7, 40.7, and 51.7 °C, respectively. These results aligned with the CD analysis, although some differences were noted due to the analysis instruments. These finding further confirmed that the thermal stability of the triple helical structure in collagen could be enhanced by N-terminal polymer modification at low pH, and longer PAA chains were more beneficial for the thermal stability of collagen.

Fig. 7
figure 7

A The CD value at 220 nm-temperature curves of different collagens, SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l). B The derivative thermal denaturation curves of different collagens, SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l). C DSC thermograms of SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l)

Full size image

3.6 Critical pH of SPSC-PAA

As a pH responsive polymer, PAA modification of collagen improves its pH sensitivity. Here, the phase transition behavior of SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) was evaluated. This was done by turbidity measurement in the pH range of 2–13, focusing on the critical pH at which PAA conformation starts to change. As shown in Fig. 8A, the turbidity of the ASC and SASC systems remained almost unchanged with the increase in pH value, indicating that neither of them possesses pH-responsive phase transition properties. Conversely, a rapid decrease in turbidity was observed for SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l). This aggregation of SPSC-PAA was caused by the conformation changes of PAA from stretched at high pH to coiled at low pH. Subsequently, based on the derivative curves, the critical pH of SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) was determined to be 4.73, 4.54, and 4.26, respectively (Fig. 8B). The differences in pH-responsive turbidity between SPSC-PAA and ASC are shown in Fig. 8C, D. The producibility of the critical pH differences of SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) might be explained by the different polymer chain lengths.

Fig. 8
figure 8

A The turbidity-pH curves of SPSC-PAA(s), SPSC-PAA(m), SPSC-PAA(l), ASC, and SASC in the pH range of 2–13. B The critical pH of SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) was obtained from the derivative curves of turbidity-pH in (A). C, D The images show the turbidity change for SPSC-PAA(m) (C) and ASC (D) at pH 4 and 8, respectively

Full size image

3.7 Isoelectric point of SPSC-PAA

As an amphipathic biomacromolecule, the solubility of collagen is closely related to its isoelectric point. Therefore, the impact of PAA modification and PAA chain length on collagen hydrophilicity was explored through isoelectric point analysis. The isoelectric point of a sample is the pH when its zeta potential becomes 0 [15]. The isoelectric point titration curves of ASC, SASC, SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l) are shown in Fig. 9. After succinylation, the isoelectric point of ASC (6.8) decreased to 4.8 (SASC), which is consistent with previous reports [28]. Succinylation of amino groups in collagen alters its net surface charge, as amino groups are replaced by carboxyl groups [13]. When PAA, containing a large number of carboxyl groups, was introduced at the N-terminal of SPSC, the increase in negatively charged carboxyl groups, resulted in a decrease in the isoelectric point of SPSC-PAA. The isoelectric point of SPSC-PAA(s) was 4.75, similar to the 4.8 of SPSC. However, the isoelectric points of SPSC-PAA(m) and SPSC-PAA(l) decreased to 4.30 and 3.86, respectively. These results suggest that the isoelectric point of SPSC-PAA is affected by the polymer chain length.

Fig. 9
figure 9

Isoelectric point titration curves of collagens, SPSC-PAA(s), SPSC-PAA(m), and SPSC-PAA(l)

Full size image

4 Conclusions

The SPSC-PAA bioconjugates with different PAA chain lengths were prepared by the site-specific N-terminal α-amino modification strategy. The TNBS assay results confirmed that the modification rate of amino groups in SPSC-PAA reached up to 97%. All N-terminal α-amino groups in collagen underwent modification. This modification did not destroy the triple helical structure of collagen. The thermal stability of SPSC-PAA showed pH responsiveness, as indicated by the increased denaturation temperature under acidic conditions compared to alkaline conditions. The pH-responsive mechanical property was further confrmed by rheology. Longer PAA chains were found to be more beneficial for the thermal stability of SPSC-PAA. The critical pH and isoelectric point of SPSC-PAA decreased with the increasing chain length of PAA. This work improves the understanding of the impact of the N-terminal modification of collagen on its properties. Such insights are expected to aid in the design of collagen bioconjugates.

Availability of data and materials

Not applicable.

Abbreviations

ASC:

Tropocollagen with terminal telopeptides

PSC:

Atelocollagen without terminal telopeptides

SASC:

Succinylated ASC

SPSC:

Succinylated PSC

SPSC-ini:

ATRP initiators modified SPSC

SPSC-PAA:

Succinylated collagen-PAA conjugate

AA:

Acrylic acid

PAA:

Polyacrylic acid

TNBS:

Tri-nitro benzene sulfonic acid

HMTETA:

1,1,4,7,10,10-Hexamethyltriethylenetetramine

Gly:

Glycine

Pro:

Proline

Hyp:

Hydroxyproline

PNIPAAm:

Poly(N-isopropylacrylamide)

ATRP:

Atom transfer radical polymerization

Mn :

The number average molecular weight

PDI:

Polydispersity index, Mw/Mn

GPC:

Gel permeation chromatography

CD:

Circular dichroism

References

  1. Zhang X, Xu S, Shen L, Li G. Factors affecting thermal stability of collagen from the aspects of extraction, processing and modification. J Leather Sci Eng. 2020;2:19.

    Article  Google Scholar 

  2. Yan X, Chen Y, Dan W, Dan N, Li Z. Bletilla striata polysaccharide modifed collagen fIber composite sponge with rapid hemostasis function. Collagen and Leather. 2022;4:5.

    CAS  Google Scholar 

  3. Li X, Li M, Guo J, Liu X, Liao X, Shi B. Collagen peptide provides Streptomyces coelicolor CGMCC 4.7172 with abundant precursors for enhancing undecylprodigiosin production. J Leather Sci Eng. 2021;3:17.

    Article  CAS  Google Scholar 

  4. Lee CH, Singla A, Lee Y. Biomedical applications of collagen. Int J Pharm. 2001;221:1–22.

    Article  CAS  Google Scholar 

  5. Gelse K, Pöschl E, Aigner T. Collagens-structure, function, and biosynthesis. Adv Drug Deliv Rev. 2003;55:1531–46.

    Article  CAS  Google Scholar 

  6. Riemschneider R, Abedin MZ. Pepsin-solubilized collagen from cow placenta. Angew Makromol Chem. 1979;82:171–86.

    Article  CAS  Google Scholar 

  7. Brodsky B, Persikov AV. Molecular structure of the collagen triple helix. Adv Protein Chem. 2005;70:301–39.

    Article  CAS  Google Scholar 

  8. Usha R, Ramasami T. Influence of hydrogen bond, hydrophobic and electrovalent salt linkages on the transition temperature, enthalpy and activation energy in rat tail tendon (RTT) collagen fibre. Thermochim Acta. 1999;338:17–25.

    Article  CAS  Google Scholar 

  9. Xu C, Xiao X, Hu W, Zhu L, Kou H, Zhang J, Wei B, Wang H. Ultrahigh pressure field: a friendly pathway for regulating the cellular adhesion and migration capacity of collagen. Int J Biol Macromol. 2023. https://doi.org/10.1016/j.ijbiomac.2023.127864.

    Article  Google Scholar 

  10. Zhang ZK, Li GY, Shi B. Physicochemical properties of collagen, gelatin and collagen hydrolysate derived from bovine limed split wastes. J Soc Leather Technol Chem. 2006;90:23–8.

    Google Scholar 

  11. Sripriya R, Kumar R, Balaji S, Kumar MS, Sehgal PK. Characterizations of polyanionic collagen prepared by linking additional carboxylic groups. React Funct Polym. 2011;71:62–9.

    Article  CAS  Google Scholar 

  12. Zhang J, Sui P, Yang W, Shirshin EA, Zheng M, Wei B, Xu C, Wang H. Site-specific modification of N-terminal α-amino groups of succinylated collagen. Int J Biol Macromol. 2023;225:310–7.

    Article  CAS  Google Scholar 

  13. Kim SH, Lee JH, Yun SY, Yoo JS, Jun CH, Chung KY, Suh H. Reaction monitoring of succinylation of collagen with matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrometr. 2000;14:2125–8.

    Article  CAS  Google Scholar 

  14. Zhang Z, Liu W, Li D, Li G. Physicochemical properties of succinylated calfskin pepsin-solubilized collagen. Biosci Biotechnol Biochem. 2007;71:2057–60.

    Article  CAS  Google Scholar 

  15. Wang W, Shu F, Pan L, Huang S, Tu X, Li P, Li S, Li Y, Xu C, Sun Y, Zhang J, Wang H. Impact of grafting density on the self-assembly and hydrophilicity of succinylated collagen. Macromol Res. 2020;28:636–43.

    Article  CAS  Google Scholar 

  16. Zhang J, Deng F, Liu W, Huang Y, Tu X, Kou H, He L, Wei B, Xu C, Wang H. Temperature-responsive collagen–PNIPAAm conjugate: preparation and fibrillogenesis. New J Chem. 2020;44:21261–70.

    Article  CAS  Google Scholar 

  17. Chen L, Zeng Z, Li W. Poly(acrylic acid)-assisted intrafibrillar mineralization of type I collagen: a review. Macromol Rapid Comm. 2023;44:2200827.

    Article  CAS  Google Scholar 

  18. Hou X, Liu Y, Dong H, Yang F, Li L, Jiang L. A pH-gating ionic transport nanodevice: asymmetric chemical modification of single nanochannels. Adv Mater. 2010;22:2440–3.

    Article  CAS  Google Scholar 

  19. Wang H, Liang Y, Wang H, Zhang H, Wang M, Liu L. Physical-chemical properties of collagens from skin, scale and bone of grass carp (Ctenopharyngodon Idellus). J Aquat Food Prod T. 2014;23:264–77.

    Article  CAS  Google Scholar 

  20. Cummings C, Murata H, Koepsel R, Russell AJ. Tailoring enzyme activity and stability using polymer-based protein engineering. Biomaterials. 2013;34:7437–43.

    Article  CAS  Google Scholar 

  21. Tronci G, Russell SJ, Wood DJ. Photo-active collagen systems with controlled triple helix architecture. J Mater Chem B. 2013;1:3705–15.

    Article  CAS  Google Scholar 

  22. Shu F, Dai C, Wang H, Xu C, Wie B, Zhang J, Xu Y, He L, Li S. Formation, stability and self-assembly behaviour of the collagen-like triple helix confirmation: the role of Ser. Ala and Arg/Glu ChemistrySelect. 2019;4:13370–9.

    CAS  Google Scholar 

  23. Xiang L, Cui W. Biomedical application of photo-crosslinked gelatin hydrogels. J Leather Sci Eng. 2021;3:3.

    Article  CAS  Google Scholar 

  24. Zhang J, Tu X, Wang W, Nan J, Wei B, Xu C, He L, Xu Y, Li S, Wang H. Insight into the role of grafting density in the self-assembly of acrylic acid-grafted-collagen. Int J Biol Macromol. 2019;128:885–92.

    Article  CAS  Google Scholar 

  25. Mils CA, Bailey AJ. Thermal denaturation of collagen revisited. Proc Indian Acad Sci-Chemical. 1999;111:71–80.

    Article  Google Scholar 

  26. Bo Q, Zhao Y. Double-hydrophilic block copolymer for encapsulation and two-way pH change-induced release of metalloporphyrins. J Polym Sci Part A Polym Chem. 2006;44:1734–44.

    Article  CAS  Google Scholar 

  27. Li G, Song S, Guo L, Ma S. Self-assembly of thermo- and pH-responsive poly(acrylic acid)-b-poly(N-isopropylacrylamide) micelles for drug delivery. J Polym Sci Part A Polym Chem. 2010;46:5028–35.

    Article  Google Scholar 

  28. Li C, Tian H, Duan L, Tian Z, Li G. Characterization of acylated pepsin-solubilized collagen with better surface activity. Int J Biol Macromol. 2013;57:92–8.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This study was financially supported by the National Natural Science Foundation of China (Nos. 22378320, 22178277, 21706201), Knowledge Innovation Program of Wuhan-Basi Research, China (No. 2023020201010148), and Application Foundation Frontier Project of Wuhan Science and Technology Bureau, China (No. 2019020701011478).

Author information

Authors and Affiliations

  1. School of Chemical and Environmental Engineering, Wuhan Polytechnic University, Wuhan, Hubei, China

    Juntao Zhang, Yang Liu, Haofei Xu, Peishan Sui, Mingming Zheng, Benmei Wei & Chengzhi Xu

  2. College of Life Science and Technology, Hubei Key Laboratory of Quality Control of Characteristic Fruits and Vegetables, Hubei Engineering University, Xiaogan, Hubei, China

    Haibo Wang

  3. School of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, Hubei, China

    Tianyi Liu

  4. Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Oilseeds Processing, Ministry of Agriculture, Hubei Key Laboratory of Lipid Chemistry and Nutrition, Wuhan, Hubei, China

    Mingming Zheng

  5. Department of Physics, M. V. Lomonosov Moscow State University, Leninskie Gory 1/2, Moscow, Russia, 119991

    Evgeny A. Shirshin

Authors
  1. Juntao Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Haofei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peishan Sui
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tianyi Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mingming Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Evgeny A. Shirshin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Benmei Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chengzhi Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Haibo Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JZ: Conceptualization, Methodology, Writing, Funding acquisition, Supervision, Project administration. YL: Conducted the experiment, writing—original draft. HX: Data curation. PS: Revision. TL: Revision. MZ: Formal analysis. EAS: Revision. BW: Investigation. CX: Resources. HW: Funding acquisition, Supervision.

Corresponding author

Correspondence to Haibo Wang.

Ethics declarations

Competing interests

They were not involved in the editorial review, or the decision to publish this article. All authors declare that there are no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, J., Liu, Y., Xu, H. et al. The impact of N-terminal modification of PAA with different chain lengths on the structure, thermal stability and pH sensitivity of succinylated collagen. Collagen & Leather 6, 6 (2024). https://doi.org/10.1186/s42825-024-00148-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s42825-024-00148-8

Keywords