*2.4. HSD Interacts with Collagenase*

Collagenase enzymes are a group of metalloproteinases responsible for the degradation of collagen and may cause an ageing effect in skin when present in higher levels in extracellular matrix. Figure 3 presents suppression of fluorescence quenching—observed for collagenase (*Clostridium histolyticum*, Sigma-Aldrich) by the addition of hesperidin in different concentrations (DMSO solution). Hesperidin, as other flavonoids, can reduce the intensity of tryptophan or other fluorophores of enzyme fluorescence emissions. Hesperidin chelates zinc(II) ion, which is in the catalytic site of the enzyme, and changes the conformation of the protein. There are already various studies in which it has been proven that flavonoids have an inhibitory effect on metalloproteinase (collagenase, elastases, and hyaluronidases) by chelating their metal ions. Additionally, the temperature of the experiment was varied as to evaluate and calculate the thermodynamic values of enthalpy (ΔH) and entropy (ΔS) of collagenase–hesperidin interaction.

**Figure 3.** Illustration of fluorescence spectroscopy data: (**a**) Collagenase (*Clostridium hystolicum*) at 37 ◦C upon addition of HSD (in DMSO) at concentrations from 0.08 to 0.90 mmol L−1. (**b**) Diagram of log((F0−F)/F) in function of log[HSD].

The mechanism of fluorescence suppression can be explained through collision quenching between the excited enzyme and suppressor (*Q*), defined by bimolecular quenching constant *Kq* according to Stern–Volmer [34]. The reduction in the intensity of the fluorescence emission (*F*) in relation to the intensity observed in the absence of suppressor (*F*0) is given by Equation (1):

$$(F\_0/F) = 1 + K\_q \tau\_0[Q] = 1 + K\_S[Q] \tag{1}$$

[*Q*] is a concentration of HSD, τ0 is 10−<sup>8</sup> s, and *Kq* is the association constant, 0.29 × 10−<sup>3</sup> mol−<sup>1</sup> L, 0.32 × 10−<sup>3</sup> mol−<sup>1</sup> L, or 0.15 × 10−<sup>3</sup> mol−<sup>1</sup> L obtained for collagenase–HSD at 25 ◦C, 30 ◦C, and 37 ◦C, respectively. The active site number was estimated at n = 1.2 (1.26, 1.24, and 1.19 for collagenase–HSD interaction at 25 ◦C, 30 ◦C, and 37 ◦C, respectively). It was possible to calculate Gibbs free energy, entropy, and enthalpy, ref [35] for collagenase–HSD at 25 ◦C, 30 ◦C, and 37 ◦C, as shown in Table 1.

**Table 1.** Thermodynamic parameters of collagenase–HSD interaction.


According to the data (Table 1) and as well to Ross and Subramanian [35], the interaction between collagenase and HSD is a partial immobilization. This interaction occurs when protein and ligand are

leaving the state from which the two are separated and when they are hydrophobically associated. Further, this type of association contributes to the decrease in the ΔS and ΔG values.
