Physicochemical and Spectroscopic Characterization of Glycogen and Glycogen Phosphorylase b Complexes

Abstract

Glycogen is a natural polysaccharide used as an energy storage macromolecule. The role of glycogen metabolism in type 2 diabetes mellitus has been under investigation for several years, along with its implication in cancer and cardiovascular and neurodegenerative diseases. Previous studies using pig liver glycogen with rabbit muscle glycogen phosphorylase (RMGPb), which catalyzes the first step of glycogen degradation to glucose-1-phosphate, showed that the surface of an average glycogen molecule is covered by a total of 20 RMGPb dimeric molecules. In this work, we selected oyster glycogen (Glyc) to investigate its interaction with RMGPb by employing biophysical techniques. Dynamic, static, and electrophoretic light scattering were used to investigate the solution behaviors and structures of both the Glyc molecule itself and the formed complexes between Glyc and GPb at different mixing ratios. It was established that the interaction between oyster Glyc and RMGPb is similar to that previously reported for pig liver glycogen. Moreover, the structure of the complexed GPb was monitored by fluorescence and FTIR spectroscopy.

1. Introduction

Glycogen is a branched polymer of glucose that humans and animals use as an energy reserve. In linear chains, glucose residues connect via α-1,4 glycosidic bonds, and in order to create branch points they connect via α-1,6 glycosidic ones. Glycogen branching is a key property of glycogen as it increases its solubility and allows it to become metabolized more quickly [1,2]. Τhe role of glycogen in physiological processes has been investigated for a long time, and the literature on this research area is rather rich. However, it is only during recent years that the scientific community has started utilizing its properties as a material or nanomaterial for various applications, with a special focus towards nanomedicine [3]. The main reason for this is that there is an increasing interest in employing natural polysaccharides, including glycogen, as natural biomaterials due to their unique advantages of being biocompatible and biodegradable. Moreover, they can be chemically or biochemically modified to produce functional derivatives, which are important for various therapeutic applications [4,5]. Glycogen is rather attractive compared to other polysaccharides since it is not only biodegradable but also non-toxic. However, any chemical modification can lead to a cytotoxic and mutagenic reaction, which implies that glycogen nanoparticles require careful evaluation prior to their use [6].

The progress made so far in the development of glycogen as a functional advanced material is promising, but several key challenges need to be overcome. Studies on the in vivo fate of glycogen particles of different sizes, molecular weights, and surface functionalities, particularly with respect to the immune system and disease engagement, require further investigation [7]. With the aim of analyzing the chemistry of glycogen molecules, it is essential to define molecular mass distributions, average chain lengths, and average branching frequencies. Important insights into the glycogen structure have emerged from the chemical analysis of mammalian glycogen, suggesting that a full-size glycogen molecule would consist of a total of ~55,000 glucose residues, a molecular mass of ~107 kDa, and a diameter of ~44 nm [8]. It is highly concentrated in the liver, although skeletal muscles contain the most glycogen by weight. It is also present in lower levels in other tissues, such as the kidney, heart, astrocytes, neurons, and brain. Importantly, glycogen serves to maintain glucose homeostasis in animal and human bodies. Because of this, its metabolism is primarily regulated by insulin and glucagon and other molecules in their downstream signaling pathways. Insulin and glucagon promote glycogen synthesis and breakdown, respectively. Many pathological conditions have been associated with different points of the glycogen metabolism pathway. Diseases involving enzymes that have important roles in glycogen synthesis, degradation, and/or regulation have significant adverse effects on the body [8,9]. Finding the links between glycogen metabolism and serious diseases, such as type 2 diabetes mellitus [10], cancer [11], and cardiovascular [12,13] and neurodegenerative diseases [14], remains a challenge for the scientific community.

The development of new research tools for integrated approaches currently allows for the systematic study of enzymes (molecular targets) involved in the metabolic pathway of glycogen biosynthesis and degradation in different tissues. Of the multitude of enzymes involved in this metabolic pathway, glycogen phosphorylase (GP, EC 2.4.1.1) plays a major role, catalyzing the first stage of its intracellular degradation to glucose 1-phosphate (Glc-1-P) and then to glucose [15,16]. GP is an allosteric enzyme which exists in at least two interconvertible forms, namely GPb (inactive, T-configuration) and GPa (active, R-configuration). Its distinct binding sites have been targeted for the development of new drugs using the structure-based drug design approach [17,18]. In the liver, the main mechanism controlling glycogenolysis is the phosphorylation of GPb → GPa, and according to previous studies, glucose inhibits the action of GP by synergistically acting with insulin to reduce degradation while increasing glycogen synthesis.

Previous studies using pig liver glycogen with rabbit muscle GPb (RMGPb) explored the complex formation between the two, and the kinetics of their interaction were monitored by the differences observed in the intensity of scattered light upon enzyme binding to glycogen. The equilibrium dissociation constants of the complexes and the adsorption capacity of glycogen were determined by sedimentation methods. The results show that the surface of an average glycogen molecule is covered by a total of 20 GPb dimeric molecules [19]. In this work, we investigate the solution properties of oyster Glyc and its complexation process with RMGPb as well as the biophysical properties of the formed complexes by means of dynamic, static, and electrophoretic light scattering (DLS, SLS, and ELS) techniques. Oyster Glyc was selected due to its high purity (<1% reducing sugars) and appropriateness for various applications concerning life sciences, biopharma, metabolomics, pharmaceuticals, etc. We present the interaction of the oyster Glyc-GPb system by focusing on the physicochemical properties and dynamic character of this interaction, paving the way for further studies involving natural polysaccharides as carriers. Furthermore, we report, for the first time, DLS measurements performed in “real-time” and during titration experiments, aiming to investigate the kinetics of the interaction between the two components, Glyc and GPb. Finally, spectroscopic methods like fluorescence spectroscopy (FS) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) are also used to assess the tertiary and secondary structure, respectively, of the enzyme after complexation [20,21].

2. Materials and Methods

2.1. Materials

All chemicals were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). BES buffer (99.5%), sodium chloride (NaCl, 99%), hydrogen chloride solution (HCl, 37%), ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA 98.5%), 1,4-dithiothreitol (DTT), sodium azide (NaN₃, 99.5%), ethanol 100%, adenosine monophosphate (AMP), and toluene were used without any further purification.

2.2. Sample Preparation

Protein. RMGPb was isolated from rabbit skeletal muscle according to a variation in the protocol of the Fischer and Krebs method in which β-mercaptoethanol replaces L-cysteine in all purification steps [22]. The concentration of purified RMGPb was measured spectrophotometrically by the Beer–Lambert law using absorbance at 280 nm (the calculated molar absorption coefficient was 13.2 (mg/mL)⁻¹ cm⁻¹ at 280 nm for a 1% wt. protein solution) [23]. RMGPb was dialyzed against BES buffer and was treated with charcoal to remove the remaining nucleotides and other impurities prior to complexation.

Glycogen. The preparation of glycogen from oyster (glycogen from oyster type II, Sigma-Aldrich G8751) also included treatment with charcoal to remove free glucose, residues containing glycans, and other biomolecules that react with the analytical reagents. The treated glycogen was then lyophilized and dialyzed against deionized water to a final concentration of 10% w/v. After treatment, the glycogen was stored at −19 °C prior to use. For the investigation of its solution properties, a 10 mg/mL stock solution was prepared by direct dissolution in water for injection (WFI) containing 200 ppm NaN₃ (so as to prevent bacterial growth). Subsequently, appropriate dilutions were performed in order to produce a range of solutions with concentrations of 1, 2.5, 5, and 7.5 mg/mL.

Preparation of Complex Solutions. The solution behaviors and structures of the formed complexes between oyster glycogen (Glyc) and GPb at different mixing ratios were investigated. All solutions were prepared using N,N-bis(2-hydroxyethyl)-2-amino ethanesulfonic acid buffer at pH 6.8 in the presence of 1 mM AMP. For the preparation of the different complex solutions, 1 mL of a 1 mg/mL Glyc solution was mixed with 0.1, 0.5, 1, 1.5, 2, 3, or 4 mL of a 1 mg/mL GPb solution, and then all solutions were diluted with BES buffer to a final volume of 5 mL. This way, the concentration of glycogen was kept constant at 0.2 mg/mL throughout the series of complex solutions, while that of the enzyme ranged from 0.02 to 0.8 mg/mL, thus changing the molar ratio of the two components. Moreover, the pH of the complex solutions was considered equal to that of the BES buffer (i.e., pH = 6.8). The corresponding mixing/molar ratios between glycogen and the enzyme for each complex solution are summarized in

Table 1. The compositions of the different Glyc + GPb complex solutions prepared for this study. The concentration of Glyc is equal to 0.2 mg/mL for all complex solutions.

Name	Enzyme Concentration CGPb (mg/mL)	Total Complex Concentration CTot (mg/mL)	Enzyme/Glycogen Molar Ratio molGPb/molGlyc
Comp(1 + 0.1)	0.02	0.22	1.8
Comp(1 + 0.5)	0.1	0.3	9
Comp(1 + 1)	0.2	0.4	18
Comp(1 + 1.5)	0.3	0.5	27
Comp(1 + 2)	0.4	0.6	36
Comp(1 + 3)	0.6	0.8	54
Comp(1 + 4)	0.8	1	72

. All solutions were left to stand overnight for better equilibration.

In order to investigate the kinetics of the complexation process between the two components, two different types of experiments were conducted. The first one involved the “real-time” measurement of the complex solution directly after mixing as a function of time, while the second one probed the complexation as a titration procedure, i.e., after consecutive additions of small aliquots of the enzyme solution to the glycogen one. More specifically, for the real-time experiment, 1 mL of a 1 mg/mL glycogen (Glyc) solution was mixed with 1 mL of an either 1.5 or 0.75 mg/mL RMGPb solution directly in the DLS glass cell and then measured continuously every ~1 min for ~25 min. In a similar manner, for the titration experiment, in 1 mL of a 1 mg/mL glycogen (Glyc) solution, a total of 1 mL of an either 1.5 or 0.75 mg/mL GPb solution was added in aliquots of 100 μL (i.e., 10 additions in total) directly in the DLS glass cell, and the solution was left to equilibrate for 5 min after each addition before measurement. The concentration of the GPb enzyme, 1.5 or 0.75 mg/mL, was chosen so the molar ratio of the two components, mol_GPb/mol_Glyc, was equal to 27 or 13.5, respectively. All solutions were prepared with N,N-bis(2-hydroxyethyl)-2-amino ethanesulfonic acid BES buffer (10 mM BES, 0.1 mM EDTA, 0.5 mM DTT, 0.01% NaN₃) at pH 6.8 in the presence of 1 mM AMP.

2.3. Methods

Dynamic and Static Light Scattering (DLS and SLS). DLS measurements were performed on an ALV/CGS-3 compact goniometer system (ALVGmbH, Hessen, Germany) equipped with an ALV-5000/EPP multi-tau digital correlator, a He-Ne laser (λ = 632.8 nm), and an avalanche photodiode detector. All sample solutions were filtered through 0.45 μm hydrophilic PVDF syringe filters before measurement to remove any dust particles or large aggregates. The samples were loaded into standard soda–lime glass dust-free cylindrical cells that were 1 cm width, and measurements were performed at a series of angles in the range of 45–135°.

Scattered light intensity autocorrelation functions $g_2\left(\tau \right)$ and field autocorrelation functions $g_1\left(\tau \right)$ were connected via Siegert relation using Equation (1):

$$g_2\left(\tau \right)-1=\beta \left|g_1\left(\tau \right)\right|$$

(1)

where $\beta$ is a normalization constant and τ is the lag time.

The characteristic relaxation time ${\tau }_{\mathrm{rel}}\left(q\right)$, which was obtained by the CONTIN analysis, can lead to the characteristic relaxation rate Γ(q) via Equation (2), which leads to the diffusion coefficient D via Equation (3).

$$\Gamma \left(q\right)=1/{\tau }_{\mathrm{rel}}\left(q\right)$$

(2)

$$\Gamma =D{q}^2$$

(3)

Using the Stokes–Einstein equation [24], parameters such as the hydrodynamic radius (R_h) of the particles under study can be calculated as shown in Equation (4):

$$R_{\mathrm{h}}=k_{\mathrm{B}}T/6\pi \eta D_{\mathrm{T}}$$

(4)

where k_B is the Boltzmann constant, η the solution’s viscosity, and D_T is the translational diffusion coefficient [25,26].

It should be noted that for the measured scattered intensity values, there is a standard deviation of approximately 1–2%, while for the calculated R_h values, the corresponding standard deviation is about 5%.

Multiangle static light scattering (SLS) measurements were obtained by the Zimm method using Equation (5):

$$\frac{Kc}{R_{\mathsf{\theta }}}=\frac{1}{M_{\mathrm{w}}}\left(1+\frac{1}{3}{R}_{\mathrm{g}}^2{q}^2\right)+2A_{2}c$$

(5)

where M_w is the weight-averaged molecular weight, R_g is the average radius of gyration, A₂ is the second osmotic virial coefficient, c is the Glyc solution concentration, and R_θ is the corrected Rayleigh ratio (which depends on the concentration c and the magnitude of the scattering vector q), and the constant factor K is given by the following relationship:

$$K=\frac{4{\pi }^2{n}_0^2}{{\Lambda }_0^4{N}_{\mathrm{A}}}{\left(\frac{dn}{dc}\right)}^2$$

(6)

where n₀, λ₀, N_A, and dn/dc are the refractive index of the solvent, the laser wavelength in vacuum, Avogadro’s number, and the refractive index increment of Glyc with respect to the solvent, respectively. For the refractive index increment of the Glyc solutions in WFI, a value of 0.151 mL/g was used according to previous studies [27,28].

Electrophoretic Light Scattering (ELS). Zeta potential (ζ_P) measurements were obtained with a Zetasizer Nano-ZS (Malvern Panalytical Ltd., Malvern, United Kingdom) equipped with a He-Ne laser (λ = 633 nm) and an avalanche photodiode detector. The Henry equation in the Smoluchowski approximation was used for the zeta potential calculation. ζ_P values were determined using the Smoluchowski equation ${\zeta }_{\mathrm{P}}=4\pi \eta \nu /\epsilon$, where η is the solvent viscosity, ν is the electrophoretic mobility, and ε is the dielectric constant of the solvent, and they are reported as averages of fifty repeated measurements at a 173° scattering angle and at room temperature.

Fourier-Transform Infrared Spectroscopy (FTIR). A mid-infrared FTIR spectrometer (Equinox 55 by Bruker GmbH, Ettlingen, Germany) equipped with an attenuated total reflectance (ATR) accessory (DuraSamplIR II by SensIR Technologies, Danbury, CT, USA) with a spectral range of 5000–550 cm⁻¹ was used. Background spectra were obtained by recording the pristine and desiccated ATR diamond crystal surface in ambient air. The measurements of the complex solutions were performed in the dry state, by creating a thin film of the solution’s components on the ATR diamond crystal, after solvent evaporation in a stream of nitrogen. Specifically, a small aliquot of each solution was placed on the ATR element and dried under a N₂ flow before measurement. For each sample, the final spectrum is the average of three 100-scan measurements at a 2 cm⁻¹ resolution. The measurement of each sample was bracketed by two background spectra to allow for the elimination of H₂O vapor bands by interpolation. Moreover, the spectral contribution of the specific buffer used for the preparation of the complex solutions (BES buffer with 1 mM AMP) was suitably subtracted from the final spectra.

Fluorescence Spectroscopy. Fluorescence arises from a three-stage process occurring in specific molecules, typically polyaromatic hydrocarbons or heterocycles, referred to as fluorophores or fluorescent dyes. A fluorescent probe is a specially designed fluorophore that reacts to a specific stimulus or targets a particular region within a biological specimen [29]. The steady-state fluorescence spectra of the tryptophan residues of the neat and complexed RMGPb were recorded with a double-grating excitation and a single-grating emission spectrofluorometer (Fluorolog-3, model FL3-21, Jobin Yvon-Spex, Horiba Ltd., Kyoto, Japan) at room temperature. The excitation wavelength used was λ = 295 nm, and the emission spectra were recorded in the region of 315–500 nm, with an increment of 1 nm, using an integration time of 0.5 s. Slit openings of 1 nm were used for both the excitation and the emitted beam. Under the employed experimental conditions, fluorescence from the tryptophan residues of GPb was observed and utilized to extract information about the conformation of the enzyme, while the free glycogen solutions did not show any fluorescence.

3. Results

3.1. Glycogen Solution Properties

Initially, we investigated the solution properties of pure Glyc by performing DLS and multiangle SLS measurements of different Glyc solutions in water (specifically WFI with 200 ppm NaN₃) at concentrations in the range of 1 to 10 mg/mL. The obtained results in regard to the values of the scattered intensity at 90°, I₉₀, as a function of the glycogen concentration, the corresponding size distribution functions, and the constructed Zimm plot are presented in Figure 1. The values for the parameters derived from the multiangle SLS measurements, that is, the weight-averaged molecular weight, M_w; the radius of gyration, Rg; and the second osmotic virial coefficient, A₂, are summarized in

Table 2. Summary of multiangle static and dynamic light scattering results for pure Glyc.

Mw (106 g/mol)	Rg (nm)	Rh (nm)	ρ = Rg/Rh	A2 (10−6 mol mL/g2)
2.6	21.7	17.7	1.23	4.5

Approximate standard deviations: M_w, 5%; R_g, 13%; R_h, 17%; A₂, 26%.

. Also included in Table 2 are the values for the corresponding average hydrodynamic radius, R_h, derived from the simultaneous dynamic measurements (i.e., independent of the concentration and scattering angle) and the characteristic ratio ρ = R_g/R_h, which provides valuable information relative to the shape, density, and overall conformation of the scattering species in the solution.

As it can be seen, Figure 1a verifies the expected linear dependency between the I₉₀ values and the concentration of the solution since the scattered intensity is proportional to the total mass of the scattering species/molecules in the solution. The corresponding SDFs shown in Figure 1b are almost identical for all different concentrations measured and reveal the presence of only one narrow peak at R_h values of about 18 nm, a size which is similar to the previously reported experimental findings concerning oyster glycogen [30,31,32,33]. Moreover, no signs of aggregation between the Glyc molecules are evidenced since no additional peaks at higher R_h values are discerned even at higher concentrations. As far as the SLS results are concerned, the calculated molecular weight for the specific Glyc sample is equal to 2.6 × 10⁶ g/mol, which corresponds to an approximate number of 14,400 glucose units. This M_w value is somewhat lower than the ones found in the literature for oyster glycogen [31,32,34,35,36], which are in the range of 5 to 7 × 10⁶ g/mol. This fact could be correlated to the particular cleaning protocol/charcoal treatment implemented in this study. Regarding the size and shape of the Glyc molecule, the calculated R_g and R_h values are 21.7 and 17.7 nm, respectively, corresponding to a ρ ratio equal to 1.23, which coincides with the 1.225 theoretically predicted ρ value for hyperbranched structures [27,35]. Finally, the second virial coefficient A₂ exhibits a molecular weight dependence expressed by a scaling law of the form $A_2\propto M_{\mathrm{w}}^{a_{A_2}}$, which, in this case, results in an $a_{A_2}$ exponent value equal to −0.83, which is also indicative of hyperbranched spherical architectures [27,35].

3.2. Glycogen–Glycogen Phosphorylase Complexation

The formation of glycogen/enzyme complexes through electrostatic complexation were explored by dynamic light scattering measurements (DLS). The properties of the stable complex solutions were initially examined by DLS measurements, and Figure 2 shows the obtained scattered intensity values derived from measurements at 90°, I₉₀; as a function of the GPb concentration, C_GPb; or the enzyme-to-glycogen molar ratio, mol_GPb/mol_Glyc. More specifically, Figure 2 includes both the actual intensity values obtained from the measurement of each complex solution sample (i.e., as measured) as well as the corrected intensity values derived from the measured ones after subtracting the corresponding contributions of glycogen and the enzyme according to their relative concentration. Note that the I₉₀ value at C_GPb = 0 for the as-measured data (i.e., square symbols in Figure 2) corresponds to that of a free glycogen solution at the same concentration as in the complexes (C_Glyc = 0.2 mg/mL). The observed gradual increase in I₉₀ values, which is also evident after the correction for the contribution of the constituents, serves as a direct proof of complexation and complex formation since the scattered intensity is proportional to the mass of the scattering species in the solution.

Regarding the corresponding sizes, the solutions of the complexes exhibit one main peak in all cases with R_h values of about 18 nm, as observed in the obtained size distribution functions shown in Figure 3. Note that the free glycogen solution also exhibits a similar peak around 18 nm, as also seen in Figure 1b. As the concentration of phosphorylase increases, a broadening of the main peak of the complex solutions can be observed, but the mean size remains practically the same. One possible explanation for this observation is that the size of the complexes is mostly determined by the size of the glycogen molecules. This may further indicate that glycogen molecules may be deformed to some extent and engulf the enzyme molecules upon interaction. Quite interestingly, for complex solutions with C_GPb ≥ 0.2 mg/mL, a second small peak with a size of about 3.5 nm is observed. This peak apparently indicates the presence of uncomplexed phosphorylase in the corresponding solutions of the complexes, thus suggesting that the interaction capacity between the two components is rather limited. In other words, each glycogen molecule can bind a specific finite number of phosphorylase enzyme molecules, and as the enzyme concentration increases, an excess of GPb molecules is observed. At the same time, the contribution of the GPb peak at 3.5 nm to the overall distribution seems to increase for C_GPb values up to 0.4 mg/mL, providing evidence for the increase in uncomplexed GPb (Figure 3b), while for higher C_GPb values (≥ 0.6 mg/mL), no significant changes are observed. This increase in the uncomplexed GPb at higher enzyme concentrations or equivalent molar ratios could be an indication that the maximum interaction capacity between the two components was reached for mol_GPb/mol_Glyc ≥ 36.

Another physicochemical characteristic of the formed complexes is their effective charge, which was acquired via electrophoretic light scattering (ELS) measurements, yielding the zeta potential ζ_P values presented in Figure 4 in an identical manner to the previous DLS results. The observed decrease in the absolute value of the zeta potential as the concentration of the enzyme becomes higher is an indication of the charge neutralization that occurs due to complexation. Moreover, for C_GPb ≥ 0.4 mg/mL (or mol_GPb/mol_Glyc ≥ 36), the zeta potential of the complexes seems to reach a plateau value, similar to that of neat GPb, which was measured at about −12 ± 5 mV. According to these observations, the proposed scenario is that each spherical glycogen molecule binds in its periphery many GPb molecules, resulting in complexes that can be described as glycogen molecules externally decorated with several phosphorylases.

3.3. Glycogen–Glycogen Phosphorylase Complexation Kinetics

In an attempt to examine, in more detail, the kinetics of the complexation process between glycogen and glycogen phosphorylase, two different types of time-dependent measurements/experiments were designed and executed. Firstly, a real-time measurement was carried out in which the two components (Glyc and GPb) were mixed directly in the DLS cell followed by consecutive measurements (at a 90° angle) as a function of time. The second type of experiment was performed in a titration manner, meaning that small aliquots of the GPb solution were gradually added to that of Glyc, once again directly in the DLS cell, and after 5 min of equilibration, the measurement for each addition was performed. For both set-ups, 1 mL of a 1 mg/mL Glyc solution was initially placed in the DLS cell, and a total of 1 mL of a 0.75 or 1.5 mg/mL GPb solution was subsequently added either directly (real time experiment) or in 100 μL aliquots (titration experiment). The two different concentrations of the enzyme correspond to a molar ratio (mol_GPb/mol_Glyc) of 13.5 or 27, which are both below the limit of the maximum interaction capacity, as established from the previously obtained results for the complexes.

For the real-time experiment, the measured I₉₀ values (corrected for the contribution of the constituents) for the two different mol_GPb/mol_Glyc ratios are presented in Figure 5a as a function of time. As it can be seen, although there are some fluctuations, the scattered intensity values or, equivalently, the mass of the complexes remains practically constant throughout the entire timescale of the measurement. Apparently, the complexation process between Glyc and GPb molecules occurs too fast to be detected under specific experimental conditions. Nevertheless, it is worth noting that the overall intensity values are higher for mol_GPb/mol_Glyc = 27, thus verifying the increase in the mass of the complexes as the enzyme content increases, which is in accordance with the previous results. The same observation also stands for the corresponding sizes of the complexes, as evidenced from the R_h values shown in Figure 5b derived from the corresponding size distribution functions (shown in Figure S1). For both molar ratios, no significant changes are observed in the size of both peaks, while the main peak (R_h2) which indicates the size of the complexes seems somewhat larger overall (about 20 nm) at mol_GPb/mol_Glyc = 27 than at mol_GPb/mol_Glyc = 13.5 (about 18 nm). This finding is in accordance with the notion that at higher molar ratios, each glycogen molecule interacts with a larger number of phosphorylases.

Since the real-time monitoring of the kinetics of the complexation process proved to be unattainable, a titration experiment was conceptualized, aiming to determine whether the decrease in the number of phosphorylase molecules interacting with glycogen would affect their complexation. In this case, the experimental procedure involved the addition of small aliquots (100 μL) of the GPb solution into the DLS cell that contained the Glyc solution, as described above, and the measurement was performed five minutes after each addition of the enzyme to ensure the equilibration of the system. The same concentration of the phosphorylase solution was used, either 0.75 or 1.5 mg/mL, so the final (after the addition of the whole 1 mL of the enzyme solution) molar ratio corresponded to mol_GPb/mol_Glyc = 13.5 or 27. Figure 6 presents the results of this measurement with regard to the corrected I₉₀ values, along with the R_h values derived from the corresponding size distribution functions (shown in Figure S2), as a function of the mol_GPb/mol_Glyc molar ratio for the two different values of C_GPb used. As it can be observed, the mass was mainly larger (I₉₀ values), and to a lesser extent, the size (R_h2 values) of the complexes formed at higher C_GPb values but with the same molar ratio. As it can be observed, the mass (I₉₀ values) of the complexes formed at higher C_GPb values but with the same molar ratio is larger, although their corresponding sizes (R_h2 values) are similar. This observation is directly correlated to the interaction degree between Glyc and GPb molecules in the sense that when the concentration of GPb is higher, each Glyc interacts with a higher number of enzyme molecules, thus forming complexes with an increased mass, although the molar ratio between the two components is the same. One logical explanation for this finding is that after the Glyc molecule has interacted with a number of GPb molecules, its ability to further bind additional ones is significantly hindered, most probably due to conformational restrictions. In other words, it is difficult for an already formed Glyc + GPb complex to accommodate additional enzyme molecules, and this affects the structure of the resulting complexes. Therefore, the complexes formed at C_GPb = 0.75 mg/mL and the same mol_GPb/mol_Glyc value have a lower mass than the ones at a higher C_GPb since they correspond to double the number of titrations. At the same time, the size of the complexes is mostly determined by the size of the Glyc molecule and therefore does not show significant differences independently of the number of phosphorylases bound per glycogen. One final remark here is the presence of free/unbound GPb (R_h1 peak), which is barely discerned at lower concentrations of the enzyme (see Figure S2).

3.4. The Enzyme Structure within the Complexes

The structure of a protein/enzyme and its configuration in space are the main properties that determine its function. For this reason, in most biochemical and biotechnological applications where proteins are involved, the control of structural conservation is of paramount importance. Therefore, it is necessary to investigate the overall structure of glycogen phosphorylase in the complexes using fluorescence and mid-infrared spectroscopic techniques, which detect the tertiary and secondary structures of the enzyme molecule, respectively.

Amongst the three fluorescent amino acid constituents of proteins, tryptophane (Trp) is the most abundant. The photophysical properties of Trp are highly sensitive to its local environment; thus, the corresponding emission spectrum can provide very important information about changes in the polarity of the surroundings of Trp residues and, accordingly, in the structure of the whole protein molecule. The other two amino acids are phenylalanine (Phe) and tyrosine (Tyr), with the first one having very low intrinsic fluorescence, while the emission of the second one is often quenched in native proteins, probably due to its interaction with the peptide chain or through energy transfer to Trp [37].

The tryptophane emission spectra (excitation at 295 nm) of the solutions of the Glyc + GPb complexes were recorded to probe the structure of the complexed enzyme. The resulting spectra are shown in Figure 7, where the corresponding spectrum of free/uncomplexed GPb, along with that of free glycogen (which confirms the absence of fluorescence) are also included for comparison. All spectra exhibit the characteristic peak of tryptophane with a maximum at about 350 nm, while the intensity of the peak is directly proportional to the concentration of GPb in the solution (for this reason, the fluorescence spectra of the solutions of the complexes at C_GPb = 0.4 and 0.1 mg/mL are directly compared with the corresponding spectra of neat GPb at concentrations of 0.5 and 0.1 mg/mL, as shown in Figure S3). Since there are no significant changes in the tryptophane peak position for the complexed enzyme, it can be concluded that GPb is not subjected to substantial conformational changes when it interacts with glycogen.

Additionally, infrared (IR) spectroscopy measurements were performed for the determination of the secondary structure of the complexed enzyme. Figure 8 presents the obtained FTIR-ATR spectra of the Glyc + GPb complexes, together with the corresponding spectra of the neat enzyme and glycogen for comparison, in the Amide I and II regions. These peaks mainly represent the vibrational profile of the peptide bond and are characteristic of protein/enzyme samples. As it can be seen, the overall spectral profile (i.e., the maximum position and shape) of both Amide bands is clearly preserved in the complexes with C_GPb ≥ 0.2 mg/mL, indicating the absence of significant configurational changes [38,39]. For the complex solutions at lower C_GPb values (i.e., 0.02 and 0.1 mg/mL), both Amide bands are not so well discerned due to the low concentration of the enzyme and the contribution of the spectral characteristics of both Glyc and the used buffer (although its contribution has been subtracted from the spectra, as shown in Figure 8). Nevertheless, no significant spectral shifts that would denote the denaturation of the enzyme are observed. Thus, the results obtained from the FTIR-ATR technique also confirm that the enzyme maintains its native conformation upon complexation.

4. Conclusions

The aim of the present study was to investigate the solution properties of oyster glycogen (Glyc) and its interaction with rabbit muscle glycogen phosphorylase b (GPb) by means of light scattering techniques, since the specific system has not yet been examined from a physicochemical point of view. Overall, the results obtained confirm the known structural characteristics of the Glyc molecule, as well as the fact that oyster glycogen interacts with GPb in a similar way to that of pig liver glycogen, as previously reported in the literature [19]. More specifically, the light scattering results confirm the hyperbranched spherical architecture of Glyc while proving that the complexation between Glyc and GPb takes place, although the apparent mass and size of the formed complexes are only slightly higher than the corresponding ones of Glyc. The observed increase in the uncomplexed GPb at a higher molar ratio suggests that a maximum interaction capacity between the two components was reached for mol_GPb/mol_Glyc ≥ 36. This finding is in good agreement with the corresponding total interaction capacity of 20 GPb dimeric molecules covering each pig liver Glyc [19], demonstrating the similarities between the two Glyc-GPb systems. Moreover, the negative effective charge of the complexes decreases as a function of the GPb concentration, reaching values closer to that of the pure enzyme, indicating that the resulting complexes can be described as glycogen molecules decorated with several phosphorylases. Additionally, the fluorescence and infrared spectroscopic investigation confirmed that the structure of the enzyme remains stable even after complexation. Finally, the recording of the kinetics of the complexation process proved to be unfeasible via real-time DLS measurements, most probably because it is too fast, while it was also established that the mixing protocol of the two components (i.e., direct mixing or titration) affects the structure of the resulting complexes. As a closing remark, we hope that the different experimental measuring protocols in regard to light scattering techniques applied in the course of this study showcase the importance of these methods in the investigation of biological interactions in similar macromolecular systems, as well as the plethora of potentially attainable information.