Hyaluronidases are a group of enzymes that hydrolyze hyaluronic acid, a major component of the extracellular matrix. These enzymes play a critical role in tissue permeability, cell migration, and various physiological and pathological processes. Hyaluronidase is widely used in medical, cosmetic, and pharmaceutical applications, requiring a deep understanding of its types, mechanisms, and optimal concentrations. This article reviews the enzymology of hyaluronidase, its classification, mechanisms of action, and the importance of enzyme concentration in various applications.

Figure 1. Hyaluronidase structure (PDB code: 2PE4).

Classification of Hyaluronidases

• Hyaluronate 4-Glycanohydrolases (EC 3.2.1.35): These enzymes, commonly referred to as testicular-type hyaluronidases, are found primarily in mammalian spermatozoa, lysosomes, and the venoms of various insects and snakes. They hydrolyze the β-1,4-glycosidic linkages in hyaluronic acid, producing tetrasaccharides as end products.
• Hyaluronate 3-Glycanohydrolases (EC 3.2.1.36): These enzymes are primarily identified in leeches and certain hookworms. They degrade hyaluronic acid by cleaving the β-1,3-glycosidic bonds, also resulting in tetrasaccharides as end products.
• Hyaluronate Lyases (EC 4.2.2.1): Hyaluronate lyases, produced primarily by bacteria, degrade hyaluronic acid through a β-elimination mechanism to yield unsaturated oligosaccharides. This enzymatic activity facilitates bacterial dissemination through host tissues by degrading the extracellular matrix.

Figure 2. The three major groups of hyaluronidases. The EC numbers, catalysis, substrates, main products and sources of each hyaluronidase group are shown. (Bordon et al., 2015)

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Mechanism of Action

Hyaluronidases degrade hyaluronic acid by cleaving glycosidic bonds, leading to reduced viscosity and increased permeability of extracellular matrices. The mechanism varies based on enzyme type:

• Hydrolysis: Most mammalian hyaluronidases cleave the beta-1,4-glycosidic bonds via hydrolysis, generating oligosaccharides.
• Beta-Elimination: Bacterial hyaluronidases use a beta-elimination reaction to break down hyaluronic acid, yielding disaccharides with unsaturated bonds.
• Acidic and Neutral pH Activity: While mammalian hyaluronidases function optimally under acidic conditions (pH 4.5-5.5), bacterial hyaluronidases are often active at neutral to alkaline pH.

Figure 3. Structure of hyaluronic acid and cleavage by hyaluronidase. Hyaluronic acid is a polysaccharide that consists of the repeating disaccharide D-glucuronic acid and N-Acetyl-D-glucosamine. Hyaluronidase cleaves its β-1,4-glycosidic bonds. (Weber et al., 2019)

Importance of Hyaluronidase Concentration

The concentration of hyaluronidase significantly affects its activity and application. The appropriate enzyme concentration varies based on:

Medical Applications

• Ophthalmology: In ocular surgery, hyaluronidase enhances drug diffusion. Typical concentrations range from 15 IU/mL to 150 IU/mL. According to a 2008 study by Remy et al., the hyaluronidase dose was 75 IU/mL. Including hyaluronidase with mepivacaine for retrobulbar anesthesia during cataract surgery improved the safety of the procedure by providing complete muscle paralysis and faster attainment of full anesthesia. In 2004, Hamada et al. demonstrated that diplopia associated with peribulbar anesthesia was significantly more common when hyaluronidase (12.5 to 50.0 IU/mL) was not included in the anesthetic solution. Therefore, the addition of hyaluronidase eliminates diplopia after cataract surgery.
• Dermatology and Aesthetics: Used in dermal filler correction, hyaluronidase dissolves hyaluronic acid-based fillers. Recommended concentrations range from 10-150 IU per treated area, depending on filler type and volume. Aslam et al. (2005) demonstrated that the incorporation of hyaluronidase (75 IU/mL) into sub-Tenon's anesthesia has a marked effect on improving the immobility of the eye and eyelids (specifically the levator and orbicular muscles).
• Oncology: Hyaluronidase improves drug penetration in tumor tissues, requiring precise dosing to avoid excessive tissue degradation.

Pharmaceutical and Biotechnological Applications

• Drug Delivery Systems: Hyaluronidase facilitates subcutaneous and intradermal drug diffusion, the optimal concentration of hyaluronidase for drug delivery depends on the specific application and formulation. Studies have shown that concentrations of 0.3 mg/mL and 10 mg/mL can enhance drug release and absorption (Soundararajan et al., 2020). Some formulations use concentrations of 1,500 units of hyaluronidase per 5 mL (Murray et al., 2021).
• Tissue Engineering: Controlled degradation of hyaluronic acid is essential for scaffold remodeling, with enzyme concentrations adjusted according to the desired degradation rate.

Research Applications

• In Vitro Studies: Enzyme concentration is tailored based on experimental needs, with typical ranges of 10–500 IU/mL for cellular and molecular research.

Factors Influencing Hyaluronidase Activity

Several factors affect the enzymatic efficiency of hyaluronidase:

Case Studies

Case 1: Hyaluronidase Coated Molecular Envelope Technology Nanoparticles Enhance Drug Absorption via the Subcutaneous Route; Soundararajan et al., 2020

Subcutaneous (SC) chemotherapy offers potential advantages over intravenous administration, but is often limited by poor drug solubility and injection volume constraints. To overcome these challenges, researchers developed hyaluronidase-coated nanoparticles (HYD-NPs) that enhance drug encapsulation and absorption. Using Molecular Envelope Technology (MET), the study optimized the formulation of CUDC-101, a hydrophobic anticancer drug targeting EGFR, HER2 and HDAC. The HYD NPs demonstrated high drug loading, stability for up to 90 days, and improved pharmacokinetics in rats, doubling plasma drug exposure compared to uncoated NPs. In a murine xenograft model, HYD-coated MET-CUDC-101 NPs significantly prolonged survival from 15 to 43 days. These results highlight the potential of HYD NPs for effective SC delivery of poorly soluble chemotherapeutics.

Figure 4. Graphic abstract. (Soundararajan et al., 2020)

Case 2: Hyaluronidase Treatment of Synovial Fluid Is Required for Accurate Detection of Inflammatory Cells and Soluble Mediators; Brouwers et al., 2022

Synovial fluid (SF) is widely used for diagnostic and research purposes because it reflects the local inflammatory environment, but its viscosity and heterogeneous composition can affect analysis. This study investigated the importance of homogenizing SF prior to analysis by treating samples with hyaluronidase either before or after aliquoting. SF was collected from 29 arthritis patients and levels of cytokines, immunoglobulins, fatty acids, and oxylipins were measured by ELISA, Luminex, and LC-MS/MS. Cell populations were analyzed by flow cytometry after sequential centrifugation and enzymatic treatment. Results showed that homogenization reduced the variability of IgG, 17-HDHA, LTB4, and PGE2 levels, whereas cytokines and other fatty acids showed no difference. In addition, failure to homogenize resulted in incomplete immune cell isolation, with up to 70% of cells, particularly T and B cells, being lost in subsequent analyses. The results demonstrate that homogenization of SF samples improves the consistency of biomarker measurements and ensures accurate immune cell profiling, preventing misleading conclusions in clinical and research settings.

Figure 5. Hyaluronidase treatment lowers variability in IgG levels. A–F CXCL1, IL-6, IL-8, IL-10, TNFα, and total IgG levels measured by ELISA or Luminex. Dots are RA patients, and the OA patient is represented by squares. Each point represents one aliquot. Means with SD are shown. IL-8 and total IgG were analyzed in a separate set of 8 patients compared to CXCL1, IL-6, IL-10, and TNFα. ND not detected. AD above detection limit. G–L Coefficients of variation (CV) are depicted for each set of aliquots. Each line represents one patient. n=6–8 patients. Dots are RA patients, and the OA patient is represented by open squares Wilcoxon signed rank test was performed. *P<0.05 (Brouwers et al., 2022)

Understanding the science behind hyaluronidase, including enzyme types and optimal concentrations, is critical to maximizing its effectiveness in medical, pharmaceutical and aesthetic applications. Creative Enzymes offers high quality hyaluronidase products tailored to meet a variety of application needs. Contact us today to learn more about our hyaluronidase offerings and how they can support your needs.