Matrix metalloproteinase-2 (MMP-2), IC50 = 0.57 ± 0.02 µM
Matrix metalloproteinase-9 (MMP-9), IC50 = 0.25 ± 0.01 µM
Arginine-specific gingipain from Porphyromonas gingivalis (Arg-gingipain, RgpB), IC50 = 22.0 ± 2.2 µM, Ki = 15 µM
Lysine-specific gingipain from Porphyromonas gingivalis (Lys-gingipain, Kgp), IC50 = 13.8 ± 1.5 µM
Histatin 5 did not inhibit the serine proteases trypsin or chymotrypsin (IC50 > 50 µM for both). [1]
Histatin 5 targets the mitochondrial respiratory chain in Candida albicans, leading to inhibition of cellular respiration and induction of reactive oxygen species (ROS) formation. [2]

Histatin 5 belongs to a family of low-molecular-weight salivary proteins that are released by sublingual, parotid, and submandibular glands. Histatin 5 inhibits the host matrix metalloproteinases MMP-2 and MMP-9 with IC50s of 0.57 and 0.25 μM, respectively, using biotinylated gelatin as a substrate. Three peptides with distinct Histatin 5 sections are created and tested as MMP-9 inhibitors in an effort to identify the domain causing this inhibition. The inhibitory activities of peptides including residues 1 through 14 and 4 through 15 of Histatin 5 are significantly lower (IC50, 21.4 and 20.5 μM, respectively), whereas a peptide containing residues 9 through 22 had the same activity as Histatin 5 against MMP-9. Histatin 5 is a competitive inhibitor that only affects the Km with a Ki of 15 μM, according to kinetic studies of the inhibition of the Arg-gingipain[1]. The mitochondrial respiration process is inhibited by histatin 5.Candida albicans cells absorb the human salivary antifungal peptide Histatin 5 and bind intracellularly to mitochondria. In a dose- and time-dependent manner, histatin 5 5 suppresses both the respiration of whole blastoconidia and the respiration of isolated C. albicans mitochondria. State 2 respiration is inhibited by histatin 5 at 33 μM [2].

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Histatin 5 inhibited the gelatinolytic activity of MMP-2 and MMP-9 in a concentration-dependent manner using biotinylated gelatin as a substrate. Complete inhibition was observed at concentrations >1 µM. The IC50 for MMP-2 was 0.57 µM and for MMP-9 was 0.25 µM. [1]
The inhibitory domain of histatin 5 against MMP-9 was localized to its C-terminal region. A synthetic peptide comprising residues 9-22 (peptide 2) showed identical inhibitory activity (IC50 = 0.25 µM) to the full-length histatin 5. Peptides comprising residues 1-14 (peptide 1, IC50 = 21.4 µM) and residues 4-15 (peptide 3, IC50 = 20.5 µM) were significantly less potent. [1]
Histatin 5 inhibited the proteolytic activity of purified Arg-gingipain (RgpB) and Lys-gingipain from P. gingivalis in a concentration-dependent manner, with IC50 values of 22.0 µM and 13.8 µM, respectively. [1]
Kinetic analysis using Lineweaver-Burk plots revealed that histatin 5 is a competitive inhibitor of Arg-gingipain (RgpB), affecting only the Km (which increased with inhibitor concentration) while Vmax remained constant. The inhibition constant (Ki) was determined to be 15 µM via Dixon plot analysis. [1]
The inhibition of Lys-gingipain by histatin 5 was more complex. At 10 µM, histatin 5 acted as a noncompetitive inhibitor (Km unchanged, Vmax decreased). At 20 µM, it exhibited a mixed-type inhibition, affecting both Km (increased) and Vmax (decreased). [1]
Histatin 5 inhibited respiration of isolated C. albicans mitochondria. At a concentration of 33 µM, it inhibited state 2, state 3, and CCCP-uncoupled respiration by 60.3%, 86.0%, and 83.0%, respectively. [2]
Histatin 5 inhibited cellular respiration of intact C. albicans blastoconidia in a concentration- and time-dependent manner. Complete inhibition was observed within 5 minutes at 33 µM. [2]
Histatin 5 induced the formation of reactive oxygen species (ROS) in C. albicans blastoconidia and germinated cells, as detected by the fluorescent probe dihydroethidium. ROS formation was concentration-dependent and cell density-dependent. [2]
Histatin 5 induced ROS formation in isolated C. albicans mitochondria, which was abolished in the presence of the oxygen scavenger L-cysteine (5 mM) or the membrane-permeant superoxide dismutase mimetic TEMPO (1.5 mM). [2]
The candidacidal activity of histatin 5 was highly correlated with ROS formation. In assays where cells were treated with varying concentrations of histatin 5, the amount of ROS produced (measured fluorimetrically) correlated closely with the percentage of cell killing (determined by colony counting). In the presence of L-cysteine, both ROS formation and cell killing were prevented. [2]
In contrast, conventional respiratory chain inhibitors sodium cyanide and sodium azide did not induce ROS formation or kill yeast cells. The pore-forming antifungal peptide PGLa also did not induce ROS formation. [2]
Histatin 5 exhibited stronger candidacidal activity against logarithmic-phase C. albicans cells compared to stationary-phase cells, as assessed by colony counting assays. The intrinsic respiratory rate of stationary-phase cells was lower than that of logarithmic-phase cells. [2]

MMP-2 and MMP-9 are tested using biotinylated gelatin-coated microtiter plates as a substrate. In this assay, estimation of enzyme activity is based on the loss of bound biotin resulting from proteolytic activity against the gelatin-biotin complex adsorbed to the wells of microtiter plates. A stock solution of 5.4 μM MMP-9 is diluted to 10.8 nM in enzyme buffer consisting of 50 mM Tris-HCl (pH 7.5) containing 0.5 M NaCl and 5 mM CaCl2. The diluted enzyme is activated by adding 1 mM 4-aminophenylmercuric acetate and is further incubated at room temperature for 30 min. Histatin 5 at concentrations ranging from 0.005 to 100 μM is incubated with activated enzyme for 10 min before being added to the microtiter plates. The same procedure is carried out with peptide 1, peptide 2, and peptide 3. As a positive control, EDTA is used at 25 mM. After incubation of the appropriate inhibitor with the enzyme, the wells of a microtiter plate are filled with 50 μL of this mixture and the plate is incubated at 37°C for 2 h. Wells containing enzyme without inhibitor are used to determine maximal activity (100%). Wells containing substrate and buffer alone are used as controls, representing no activity (0%). To stop the reactions, the plate is washed three times with 200 μL of PBS containing 1% Tween 20. Subsequently, 50 μL of streptavidin-alkaline phosphatase (1:2, 500 dilution in water) is added to each well, and the plate is incubated for 15 min at 37°C. The plate is then washed four times with 200 μL of PBS-Tween, and 200 μL of pNPP dissolved in diethanolamine buffer (1 mg of pNPP per mL of buffer) is added for 20 min at 37°C. The absorbance is recorded at 405 nm using a microtiter plate reader[1].

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MMP-2/MMP-9 Inhibition Assay: Biotinylated gelatin (type I from swine skin) was coated onto 96-well microtiter plates. Pro-MMP-2 or pro-MMP-9 was activated using 1 mM APMA (4-aminophenylmercuric acetate) in an enzyme buffer (50 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl and 5 mM CaCl2). Activated enzyme (41 nM MMP-2 or 10.8 nM MMP-9) was pre-incubated with varying concentrations of histatin 5 or derived peptides for 10 minutes at room temperature. This mixture was then added to the gelatin-coated wells and incubated at 37°C for 2 hours. Proteolytic degradation of bound gelatin-biotin was stopped by washing. Remaining biotin was detected by adding streptavidin-alkaline phosphatase conjugate, followed by incubation with p-nitrophenyl phosphate (pNPP) substrate. Absorbance was read at 405 nm. Enzyme activity without inhibitor was defined as 100%. [1]
Arg-/Lys-Gingipain Inhibition Assay: The activity of purified Arg-gingipain (RgpB, 3.3 nM) or Lys-gingipain (Kgp, 4.0 nM) was measured spectrophotometrically. Enzymes were dissolved in assay buffer (0.2 M Tris-HCl, 0.1 M NaCl, 5 mM CaCl2, 10 mM L-cysteine, pH 7.6). The enzyme was pre-incubated with histatin 5 for 5 minutes before adding to the buffer containing the chromogenic substrate (80 µM BAPNA for Arg-gingipain or 80 µM Lys-pNA for Lys-gingipain). The reaction was carried out at 25°C in a cuvette, and the formation of p-nitroaniline was monitored by the increase in absorbance at 410 nm. Initial reaction velocities were calculated from the slope. [1]
Kinetic Analysis for Inhibition Type: To determine the mechanism of inhibition, enzyme activity was measured at six different substrate concentrations (ranging from 30 to 160 µM) in the absence or presence of two fixed concentrations of histatin 5. Initial velocities were plotted in Lineweaver-Burk plots (1/v vs. 1/[S]) to analyze changes in Km and Vmax. [1]
Determination of Inhibition Constant (Ki): For Arg-gingipain, Dixon plots were used. The reciprocal of reaction velocity (1/v) was plotted against histatin 5 concentration at two different fixed substrate concentrations (60 and 106 µM BAPNA). The Ki value was obtained from the intersection point of the two lines. [1]

ROS Formation Assay: C. albicans blastoconidia or germinated cells were loaded with the ROS-sensitive fluorescent probe dihydroethidium. The cells were then exposed to a dilution series of histatin 5 in 1 mM potassium phosphate buffer (pH 7.4). The kinetics of probe oxidation, indicating intracellular ROS formation, were monitored fluorimetrically over a time interval (e.g., 15 minutes). Fluorescence intensity was measured and correlated with histatin 5 concentration and cell density. [2]
Cell Viability/Killing Assay: Candidacidal activity was determined by colony counting. After exposure to histatin 5 for a specified time (e.g., 1 hour), cells were appropriately diluted and plated on agar plates. After incubation, colony-forming units were counted, and the percentage of killing was calculated relative to untreated controls. [2]

[1]. Salivary histatin 5 is an inhibitor of both host and bacterial enzymes implicated in periodontaldisease. Infect Immun. 2001 Mar;69(3):1402-8.

[2]. The human salivary peptide histatin 5 exerts its antifungal activity through the formation ofreactive oxygen species. Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14637-42.

Histidine protease 5 (Histatin 5) is a histidine-rich salivary antimicrobial peptide consisting of 24 amino acid residues (sequence: DSHAKRHHGYKRKFEHIKHHSHRGY, molecular weight 3037). [1] It is a natural component of human saliva, secreted by the parotid, submandibular, and sublingual glands. Its concentration in saliva has been reported to be nearly two orders of magnitude higher than the IC50 values of the MMP inhibitors found in this study. [1] This study suggests that histidine protease 5 has a novel biological function in the oral cavity: inhibiting host-derived matrix metalloproteinases (MMP-2 and MMP-9) and bacterial proteases associated with periodontal tissue destruction (gingival protease from Porphyromonas gingivalis). [1] The inhibition of MMPs may be related to the metal-chelating properties of histidine 5, as it contains a potential zinc-binding motif (HEXXH at residues 15–19) that is crucial for MMP activity. Peptides lacking the C-terminal region lose potent inhibitory activity, supporting the above hypothesis. [1] These findings suggest that histone 5 is part of the oral cavity’s inherent host defense system and may protect oral tissues from connective tissue damage. Furthermore, histone 5 could serve as a template for designing analogs to treat diseases involving these enzymes. [1] Histidine protease 5 is a histidine-rich cationic salivary antimicrobial peptide (sequence: DSHAKRHHGYKRKHFHEKHHSHRGY) consisting of 24 amino acid residues and a molecular weight of approximately 3037 Da. [2] The antifungal mechanism of histidine protease 5 includes: cellular uptake (possibly via receptor-mediated or potential-driven processes), intracellular transport and targeting of mitochondria, inhibition of mitochondrial respiration, induction of reactive oxygen species (ROS) formation due to abnormal electron transport, ultimately leading to oxidative damage-induced cell death. [2]
This study shows that the bactericidal activity of histone 5 differs from that of traditional respiratory inhibitors (which inhibit but do not kill fungi) and pore-forming peptides because it specifically depends on the production of reactive oxygen species (ROS) within target cells. [2]
Histone 5 is a natural component of human saliva and is considered part of the oral cavity's inherent host defense system. [2]

C135H196F3N51O35

3150.3167

3034.514

115966-68-2

Histatin 5 TFA

16132417

Typically exists as solid at room temperature

1.6±0.1 g/cm3

1.720

-13.36

51

48

105

217

6770

22

FC(C(=O)O[H])(F)F.O=C([C@]([H])(C([H])([H])C([H])([H])C([H])([H])N([H])/C(=N\[H])/N([H])[H])N([H])C([C@]([H])(C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H])N([H])C([C@]([H])(C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])O[H])N([H])C(C([H])([H])N([H])C([C@]([H])(C([H])([H])C1=C([H])N([H])C([H])=N1)N([H])C([C@]([H])(C([H])([H])C1=C([H])N([H])C([H])=N1)N([H])C([C@]([H])(C([H])([H])C([H])([H])C([H])([H])N([H])/C(=N/[H])/N([H])[H])N([H])C([C@]([H])(C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H])N([H])C([C@]([H])(C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])C1=C([H])N([H])C([H])=N1)N([H])C([C@]([H])(C([H])([H])O[H])N([H])C([C@]([H])(C([H])([H])C(=O)O[H])N([H])[H])=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@@]([H])(C([H])([H])O[H])C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])C([H])([H])C(N([H])[C@]([H])(C(=O)O[H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])O[H])=O)=O)C([H])([H])C([H])([H])C([H])([H])N([H])/C(=N/[H])/N([H])[H])=O)C([H])([H])C1=C([H])N([H])C([H])=N1)=O)=O)C([H])([H])C1=C([H])N([H])C([H])=N1)=O)C([H])([H])C1=C([H])N([H])C([H])=N1)=O)C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H])=O)C([H])([H])C([H])([H])C(=O)O[H])=O)C([H])([H])C1=C([H])N([H])C([H])=N1)=O)C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H]

KSXBMTJGDUPBBN-VPKNIDFUSA-N

InChI=1S/C133H195N51O33/c1-71(164-120(206)96(46-76-54-146-65-158-76)181-128(214)103(62-185)183-110(196)84(138)52-108(193)194)109(195)167-86(18-5-9-35-134)113(199)172-91(24-15-41-154-133(143)144)118(204)178-99(49-79-57-149-68-161-79)125(211)176-95(45-75-53-145-64-157-75)112(198)156-60-105(189)165-93(43-73-25-29-82(187)30-26-73)121(207)173-87(19-6-10-36-135)114(200)171-90(23-14-40-153-132(141)142)115(201)169-88(20-7-11-37-136)116(202)175-94(42-72-16-3-2-4-17-72)122(208)179-97(47-77-55-147-66-159-77)124(210)174-92(33-34-107(191)192)119(205)170-89(21-8-12-38-137)117(203)177-100(50-80-58-150-69-162-80)126(212)180-101(51-81-59-151-70-163-81)127(213)184-104(63-186)129(215)182-98(48-78-56-148-67-160-78)123(209)168-85(22-13-39-152-131(139)140)111(197)155-61-106(190)166-102(130(216)217)44-74-27-31-83(188)32-28-74/h2-4,16-17,25-32,53-59,64-71,84-104,185-188H,5-15,18-24,33-52,60-63,134-138H2,1H3,(H,145,157)(H,146,158)(H,147,159)(H,148,160)(H,149,161)(H,150,162)(H,151,163)(H,155,197)(H,156,198)(H,164,206)(H,165,189)(H,166,190)(H,167,195)(H,168,209)(H,169,201)(H,170,205)(H,171,200)(H,172,199)(H,173,207)(H,174,210)(H,175,202)(H,176,211)(H,177,203)(H,178,204)(H,179,208)(H,180,212)(H,181,214)(H,182,215)(H,183,196)(H,184,213)(H,191,192)(H,193,194)(H,216,217)(H4,139,140,152)(H4,141,142,153)(H4,143,144,154)/t71-,84-,85-,86-,87-,88-,89-,90-,91-,92-,93-,94-,95-,96-,97-,98-,99-,100-,101-,102-,103-,104-/m0/s1

(4S)-4-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]propanoyl]amino]hexanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]acetyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]hexanoyl]amino]-5-carbamimidamidopentanoyl]amino]hexanoyl]amino]-3-phenylpropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-5-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-carbamimidamido-1-[[2-[[(1S)-1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]-2-oxoethyl]amino]-1-oxopentan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-1-oxohexan-2-yl]amino]-5-oxopentanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)