Modifications enhancing reproducibility and sensitivity in the turbidimetric assay of hyaluronidase

Tam, Y.; Chan, E. C. S.

doi:10.1016/0167-7012(83)90040-4

Cited by 12 publications

(8 citation statements)

References 8 publications

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“…Enzyme assays. During enzyme purification and the determination of the optimum temperature for activity, enzyme activity was measured by the turbidity reduction assay (29). A 1-ml volume of substrate solution (hyaluronic acid at 70 FgIml) was incubated with 1 ml of enzyme solution in the presence of 0.05 M sodium phosphate buffer with 0.05 M NaCl (pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

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Purification and characterization of hyaluronidase from oral Peptostreptococcus species

Tam¹,

Chan²

1985

Infect Immun

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Hyaluronidase was purified to apparent homogeneity from the spent medium of Peptostreptococcus sp. strain 84H14S. The enzyme was purified 310-fold by ethanol precipitation, gel chromatography, and cation-exchange chromatography with a recovery of 42% of the original activity in the culture medium. The molecular weight of the purified enzyme was estimated to be 160,000 by gel filtration with Sephacryl S-300. Like bacterial mucopolysaccharidases of other sources, the enzyme carried out an eliminative reaction with the substrate, producing 4,5-unsaturated disaccharides as the final end products. Its optimum temperature of activity is 46°C. The purified peptostreptococcal hyaluronidase was different from previously reported bacterial hyaluronidases in several respects. It degraded hyaluronic acid rapidly and also exhibited some activity against chondroitin sulfate A and chondroitin sulfate C. The Kms for hyaluronic acid, chondroitin sulfate A, and chondroitin sulfate C were 0.14, 1.4, and 2.6 mg/ml, respectively. The specific activity of hyaluronidase was much higher than that of any previously purified mucopolysaccharidases. The Vmax against hyaluronic acid reached 400 mmol of product per min per mg of protein at 22°C. The peptostreptococcal hyaluronidase was also unique in that its optimum pH of activity was around neutrality, whereas other bacterial hyaluronidases were most active at acidic pHs.

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Section: Methodsmentioning

confidence: 99%

“…Determination of optimum temperature for activity. An enzyme solution was appropriately diluted for assay at 18, 24, 28, 32, 36, 37, 42, 46, 50, and 56°C to find the optimum temperature for activity in the turbidity reduction assay (29).…”

Section: Methodsmentioning

confidence: 99%

Purification and characterization of hyaluronidase from oral Peptostreptococcus species

Tam¹,

Chan²

1985

Infect Immun

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“…Turbidimetric analysis of filler HA hydrolysates induced by the homogenate of the red king crab hepatopancreas (HPC) showed an unexpected result (Figure 1(b)). Instead of the anticipated reduced HA capacity to form aggregates with acidic albumin ( [19] and Figure A1), the opposite effect was observed in the first 40 minutes-the reaction products of hydrolysis of the filler HA bound the protein and formed aggregates with it more effectively (optical density increases). After 40 minutes of the reaction time, the optical density in the turbidimetric samples decreased.…”

Section: Turbidimetric Analysis Of Ha-based Fillersmentioning

confidence: 93%

“…The analysis was conducted according to previously developed protocols with minor changes [18,19,21]. The content of the fillers was diluted by phosphate buffer (160 mM sodium hydroortophosphate * 12H 2 O, 39 mM sodium chloride, pH 5.55 was adjusted with hydrochloric acid) up to the concentration of 2.5 mg of HA in 1 mL.…”

Section: A Turbidimetric Methods For Determination Of Hyaluronidase Acmentioning

confidence: 99%

Perspectives on the Use of Hyaluronidase from the Red King Crab Hepatopancreas in Treating Filler Complications: In Vitro Investigations

Ponomareva¹,

Sliadovskii²,

Timchenko³

et al. 2019

Preprint

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This study focused on hydrolysis of cosmetic fillers hyaluronic acid (HA) and kinetics of the HA hydrolysis using the homogenate of the red king crab hepatopancreas. Turbidimetric analysis of the reaction mixture revealed a bell-shaped time dependence of aggregation formation. It was shown that the obtained homogenate has the similar activity to the commercially available hyaluronidase. The atomic force microscopy (AFM) examination found that the HA fillers were represented by spherical-like structures. These structures were destroyed under the action of the homogenate of the red king crab hepatopancreas. NMR of the reaction mixture showed that HA degradation lasts for some days, but a maximum rate of the reaction is detected in the first hours of incubation. The preparation with hyaluronidase activity obtained from the red king crab hepatopancreas could be used as potentially safe product for treating filler complications.

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“…Turbidimetric analysis was conducted according to previously developed protocols, with minor changes [18,19,20]. The content of the fillers was diluted by phosphate buffer (160 mM disodium phosphate and 39 mM sodium chloride; adjusted to pH 5.55 with hydrochloric acid) to a concentration of 2.5 mg/mL…”

Section: Determination Of Hyaluronidase Activity By the Turbidimetricmentioning

confidence: 99%

Peer Review #1 of "The effect of hepatopancreas homogenate of the Red king crab on HA-based filler (v0.1)"

2020

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In this study, several methods were used to analyze the hydrolysis of hyaluronic acid (HA)based cosmetic fillers by the hepatopancreas homogenate of the Red king crab. The results show that the homogenate and commercially available hyaluronidases have similar hydrolysis activities on the fillers. Atomic force microscopy (AFM) images reveal that the HA fillers consist mainly of spherical-like particles, which are converted into filamentous structures as a result of hydrolysis by the Red king crab hepatopancreas homogenate. Turbidimetric analysis of the hydrolysis process shows that HA aggregation with acidic albumin exhibits a bell-shaped dependence on reaction time. Analysis of the hydrolysis process by nuclear magnetic resonance (NMR) shows that HA degradation lasts several days. The maximum rate of the reaction is detected in the first hours of incubation. The data confirm that the purified homogenate of the Red king crab hepatopancreas exerts hyaluronidase activity on HA-based cosmetic fillers; therefore, it may be considered as a potential therapeutic agent for treating filler complications.

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Modifications enhancing reproducibility and sensitivity in the turbidimetric assay of hyaluronidase

Cited by 12 publications

References 8 publications

Purification and characterization of hyaluronidase from oral Peptostreptococcus species

Purification and characterization of hyaluronidase from oral Peptostreptococcus species

Perspectives on the Use of Hyaluronidase from the Red King Crab Hepatopancreas in Treating Filler Complications: In Vitro Investigations

Peer Review #1 of "The effect of hepatopancreas homogenate of the Red king crab on HA-based filler (v0.1)"

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