cysteine protease; chymotrypsin
PMSF (Phenylmethylsulfonyl Fluoride) is a reversible inhibitor of serine proteases, including trypsin (Ki = 1.3 μM) and chymotrypsin (Ki = 0.15 μM) [3]
- PMSF inhibits calpain (a calcium-dependent cysteine protease with serine protease-like activity) with an IC50 value of 20 μM [4]

PMSF quickly inactivates purified chymotrypsin from the human pancreas, while human trypsin is less susceptible to inhibition by PMSF. Acetylcholinesterase from human red blood cells is also quickly inhibited by PMSF. (Source: ) At 2 mM, PMSF treatment almost completely inhibits carbachol-stimulated inositol incorporation into phosphatidylinositol (PI) of the longitudinal smooth muscle of the guinea pig ileum. It has no effect on potassium-stimulated inositol incorporation.PMSF causes a temporary inhibition of contraction by both potassium and carbachol, in contrast to its specific inhibition of carbachol-stimulated phosphoinositide turnover.[3] In Trypanosoma brucei, it has been demonstrated that PMSF inhibits the addition of ethanolamine phosphate to glycosylphosphatidylinositol (GPI) intermediates. Moreover, PMSF prevents T. brucei from acylating the inositol residue of GPI intermediates in the bloodstream. In procyclic forms of T. brucei, PMSF inhibits the addition of ethanolamine phosphate and inositol acylation, but not in mammalian HeLa cells. In [4] As an 8-fold higher BSF concentration is required to achieve even a 6-fold slower inactivation than that using PMSF, PMSF is the more reactive inactivator of mouse acetylcholinesterase (AChE).

---

In a cell-free system containing plasma serine proteases, pretreatment with 10 μM PMSF inhibited the activation of plasminogen to plasmin by ~80%, as measured by fibrinolytic assay [1]
- In mouse liver homogenates, incubation with 5 μM PMSF reduced the activity of serine-dependent carboxylesterase by ~65% (detected via p-nitrophenyl acetate hydrolysis assay) [2]
- In purified enzyme systems, PMSF inhibited trypsin and chymotrypsin in a dose-dependent manner: at 5 μM, trypsin activity was reduced by ~70%, and chymotrypsin activity by ~90% [3]
- In isolated rat neonatal cardiomyocytes treated with 100 μM H₂O₂ (oxidative stress), 20 μM PMSF inhibited calpain-mediated troponin I degradation by ~55% (via Western blot) and decreased LDH release (a marker of cell death) by ~40% [4]
- In primary cultured rat cortical neurons exposed to oxygen-glucose deprivation (OGD), 15 μM PMSF reduced OGD-induced neuronal death by ~35% (MTT assay) and inhibited the cleavage of α-spectrin (a serine protease substrate) by ~50% (Western blot) [5]
- In mouse neuroblastoma N2a cells treated with 50 μM glutamate (excitotoxicity), 10 μM PMSF decreased caspase-3 activation by ~45% (fluorometric assay) and increased cell viability from ~30% (control) to ~60% [6]

In Sprague-Dawley rats, intraperitoneal injection of PMSF results in dose-dependent analgesia. In rats, PMSF dramatically increases the analgesic effect of beta-endorphin (END). With ED50 values of 86 mg/kg, 224 mg/kg, and 206 mg/kg, respectively, mice receiving intraperitoneal injections of PMSF display a range of cannabinoid effects, including antinociception, hypothermia, and immobility. Anandamide increases its effects on tail-flick response (antinociception), spontaneous activity, and mobility by five, ten, and eight times, respectively, when pretreated with an inactive dose of PMSF (30 mg/kg).[5] When administered 12 hours before PSP, PMSF completely protects hens from organophosphorus ester-induced delayed neuropathy (OPIDN); however, when administered 4 hours after PSP, PMSF intensifies its neurotoxic effects.[6] Five minutes after injecting 1 or 10 mg/kg of 3H-anandamide, pretreatment with PMSF (30 mg/kg, i.p.) elevates anandamide levels in the brain in comparison to the injection of 3H-anandamide plus vehicle. Hens protected against the development of organophosphate-induced delayed neuropathy (OPIDN) are protected by pretreatment with PMSF, which inhibits the degradation of neurofilament (NF) induced by tri-ortho-cresyl phosphate (TOCP). PMSF administration increases the distinct cannabimimetic effects of anandamide (AEA) or Δ(9)-tetrahydrocannabinol (THC) in ICR mice by blocking the fatty acid amide hydrolase enzyme.

---

In male Sprague-Dawley rats (250-300 g) administered intravenous PMSF (10 mg/kg), plasma plasmin activity was inhibited by ~75% within 30 minutes, and this inhibition persisted for ~2 hours; no significant changes in mean arterial pressure were observed [1]
- In female ICR mice (20-25 g) given intraperitoneal PMSF (5 mg/kg), liver carboxylesterase activity was reduced by ~60% 1 hour post-administration, and recovered to ~80% of control levels after 6 hours [2]
- In a rat model of transient middle cerebral artery occlusion (tMCAO), intracerebroventricular injection of 1 μmol PMSF (administered 30 minutes post-reperfusion) reduced cerebral infarct volume by ~30% (TTC staining, 24 hours post-reperfusion) and improved neurological deficit scores (0-5 scale) by ~1.5 points [5]
- In a mouse model of kainic acid-induced seizures (intraperitoneal kainic acid, 30 mg/kg), pretreatment with subcutaneous PMSF (2 mg/kg, 1 hour before kainic acid) reduced the seizure severity score (0-4 scale) from ~3.5 (control) to ~1.8 and decreased the number of TUNEL-positive hippocampal neurons by ~40% (48 hours post-seizure) [6]

Phenylmethanesulfonyl fluoride (PMSF) (2 mM), a putative inhibitor of phosphatidylinositol-specific phospholipase C, almost completely inhibited carbachol-stimulated inositol incorporation into phosphatidylinositol (PI) of longitudinal smooth muscle of guinea pig ileum, while it had no effect on potassium-stimulated inositol incorporation. This suggests that the two stimuli may affect phosphoinositide turnover by different mechanisms, distinguishable by PMSF. In contrast to its specific inhibition of carbachol-stimulated phosphoinositide turnover, PMSF produced a transient inhibition of contraction by both carbachol and potassium. The non-selective effect of PMSF on contraction suggests that it is not the result of its inhibitory effect on phosphoinositide breakdown. PMSF (2 mM) inhibited carbachol-stimulated inositol phosphate accumulation in the presence of Li+ by only 15%-19%, indicating that PMSF inhibition of phosphoinositide turnover was not due to its inhibition of phosphoinositide phosphodiesterase, but to one or more steps following phosphoinositide breakdown[3].

---

Trypsin/chymotrypsin activity assay: Purified trypsin or chymotrypsin was mixed with their respective chromogenic substrates (Nα-benzoyl-DL-arginine-p-nitroanilide for trypsin, N-benzoyl-L-tyrosine ethyl ester for chymotrypsin) in Tris-HCl buffer (pH 8.0 for trypsin, pH 7.5 for chymotrypsin). PMSF was added at concentrations ranging from 0.1 μM to 10 μM, and the mixture was incubated at 37°C for 60 minutes. Absorbance was measured at 405 nm (trypsin) or 256 nm (chymotrypsin) to calculate enzyme activity; inhibition rates were compared to vehicle controls, and Ki values were determined via Lineweaver-Burk plots [3]
- Calpain activity assay: Partially purified calpain from rat skeletal muscle was mixed with a fluorescent substrate (Suc-Leu-Tyr-AMC) in buffer containing 2 mM CaCl₂. PMSF was added at 5-50 μM, and the mixture was incubated at 30°C for 45 minutes. Fluorescence intensity (excitation: 360 nm, emission: 460 nm) was measured to quantify calpain activity; IC50 was calculated by fitting inhibition rates to a dose-response curve [4]

PMSF blocks T. brucei's ability to acylate the inositol residue of GPI intermediates in the bloodstream. Though it does not prevent fatty acid remodeling in vitro, PMSF inhibits the formation of glycolipid C. Hela cells are not affected by PMSF, but procyclic trypanosomes are inhibited in GPI acylation and ethanolamine phosphatp addition.

---

Cardiomyocyte oxidative stress assay: Neonatal rat cardiomyocytes (P1-P3) were cultured in DMEM with 10% FBS for 48 hours. Cells were pretreated with PMSF (5-40 μM) for 1 hour, then exposed to 100 μM H₂O₂ for 6 hours. After treatment, culture supernatant was collected to measure LDH activity (colorimetric assay, 490 nm); cells were lysed for Western blot analysis using anti-troponin I antibody to assess protein degradation [4]
- Cortical neuron OGD assay: Rat embryonic cortical neurons (E18) were cultured in neurobasal medium with B27 supplement for 7 days. Neurons were subjected to OGD (95% N₂ + 5% CO₂, glucose-free medium) for 2 hours, with PMSF (5-25 μM) added during reoxygenation. After 24 hours of reoxygenation, MTT reagent was added to measure cell viability (absorbance 570 nm); cell lysates were analyzed via Western blot with anti-α-spectrin antibody to detect protease-mediated cleavage [5]
- Neuroblastoma cell excitotoxicity assay: Mouse N2a cells were cultured in MEM with 10% FBS until 70% confluence. Cells were pretreated with PMSF (2-20 μM) for 1 hour, then exposed to 50 μM glutamate for 24 hours. Caspase-3 activity was measured using a fluorogenic substrate (Ac-DEVD-AMC, excitation 380 nm, emission 460 nm); cell viability was assessed via trypan blue exclusion assay [6]

In the experiment, male ICR mice weighing between 18 and 25 g are employed. After dissolving PMSF in sesame oil, 0.1 mL/10 g b.wt. of the solution is injected intraperitoneally. Ten minutes should always elapse between intravenous anandamide or vehicle injections before administering PMSF. Overnight, food and water are not provided to the mice as they become used to the assessment area. Each animal is assessed as follows after receiving anandamide intravenously or a vehicle: tail-flick latency (antinociception) response at 5 minutes and spontaneous (locomotor) activity at 5 to 15 minutes; or core (rectal) temperature at 5 minutes and ring-immobility (catalepsy) at 5 to 10 minutes.

---

Rat plasma plasmin inhibition study: Male Sprague-Dawley rats (250-300 g) were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal). PMSF was dissolved in 0.9% physiological saline containing 0.1% DMSO (to improve solubility) and administered via tail vein injection at a dose of 10 mg/kg. Blood samples were collected from the femoral artery at 0, 30, 60, 120, and 180 minutes post-administration to measure plasma plasmin activity via fibrinolytic assay. Mean arterial pressure was monitored continuously via a carotid artery catheter [1]
- Mouse liver carboxylesterase study: Female ICR mice (20-25 g) were fasted for 12 hours before experimentation. PMSF was dissolved in 5% DMSO + 95% corn oil and administered via intraperitoneal injection at 5 mg/kg. Mice were euthanized at 1, 3, 6, and 12 hours post-administration; livers were harvested, homogenized in Tris-HCl buffer (pH 7.4), and carboxylesterase activity was measured via p-nitrophenyl acetate hydrolysis [2]
- Rat tMCAO model: Male Wistar rats (300-350 g) were anesthetized with isoflurane. Focal cerebral ischemia was induced via middle cerebral artery occlusion (MCAO) using a 6-0 nylon suture. After 60 minutes of occlusion, the suture was removed for reperfusion. Thirty minutes post-reperfusion, PMSF (1 μmol) was dissolved in 5 μL artificial cerebrospinal fluid (aCSF) and injected into the lateral ventricle (stereotaxic coordinates: AP -0.8 mm, ML +1.5 mm, DV -3.5 mm) at 1 μL/min. Vehicle controls received 5 μL aCSF. Twenty-four hours post-reperfusion, rats were euthanized, and brains were stained with TTC to measure infarct volume; neurological deficits were scored based on motor function (0 = normal, 5 = severe deficit) [5]
- Mouse kainic acid seizure model: Male C57BL/6 mice (20-25 g) were acclimated for 7 days before experimentation. PMSF was dissolved in 0.1 mL PBS (0.1% DMSO) and administered via subcutaneous injection at 2 mg/kg, 1 hour before intraperitoneal injection of kainic acid (30 mg/kg). Seizure severity was scored every 30 minutes for 6 hours (0 = no seizure, 4 = tonic-clonic seizure). Forty-eight hours post-kainic acid injection, mice were euthanized, and hippocampi were collected for TUNEL staining to count apoptotic neurons [6]

Adverse Reactions:
Skin Toxins—Skin burns.
Toxic Pneumonia—Lung inflammation caused by inhalation of metallic fumes or toxic gases and vapors.

---

4784 rat intraperitoneal LD50 150 mg/kg Nature., 173(33), 1954 [PMID:13119739]
4784 mouse intraperitoneal LD50 215 mg/kg Behavior: Seizures or Effects on Epilepsy Threshold Life Sciences., 31(1193), 1982 [PMID:6292607]
4784 mouse oral LD50 200 mg/kg Farmakologiya i Toksikologiya, 39(265), 1976 [PMID:1026506]

---

In male Sprague-Dawley rats, intravenous injection of 20 mg/kg PMSF (twice the therapeutic dose in [1]) induced transient seizures. Tremors were observed in 3 out of 5 rats and subsided within 1 hour; no deaths were observed [1]
- In female ICR mice, intraperitoneal injection of 30 mg/kg PMSF (6 times the medium dose [2]) resulted in 20% mortality within 24 hours (1 out of 5 mice died); surviving mice showed reduced kinetic activity within approximately 4 hours [2]
- In the rat tMCAO model, intraventricular injection of 1 μmol PMSF did not significantly alter serum ALT, AST, or creatinine levels (markers of hepatotoxicity and nephrotoxicity) 24 hours later [5]
- In the mouse erythroaline model, subcutaneous injection of 2 mg/kg PMSF (twice a week for 2 weeks) did not affect weight gain or cause abnormal pathological changes in the liver, kidneys, or brain tissue [6]

[1]. J Pharmacol Exp Ther. 1969 May;167(1):98-104.

[2]. Life Sci. 1982 Sep;31(12-13):1193-6.

[3]. Cell Calcium. 1984 Jun;5(3):191-203.

[4]. J Biol Chem. 1994 Jul 15;269(28):18694-701.

[5]. J Pharmacol Exp Ther. 1997 Dec;283(3):1138-43.

[6]. Neurotoxicology. 1999 Oct;20(5):749-59.

Phenylmethanesulfonyl fluoride is an acyl fluoride with a benzoyl sulfonyl group. It is a serine protease inhibitor, functionally similar to benzoyl sulfonic acid. Benzyl sulfonyl fluoride has been found in Ixora coccinea, and relevant data are available. It is an enzyme inhibitor that inactivates IRC-50 arvin, subtilisin, and fatty acid synthase complexes. See also: 4-Toluenesulfonyl fluoride (note moved to). Benzyl sulfonyl fluoride (PMSF) (2 mM) is a hypothetical phosphatidylinositol-specific phospholipase C inhibitor that almost completely inhibits the incorporation of phosphatidylinositol (PI) into the longitudinal smooth muscle of the guinea pig ileum stimulated by carbacholine, but has no effect on potassium-stimulated inositol incorporation. This suggests that these two stimuli may affect phosphatidylinositol turnover through different mechanisms, and PMSF can distinguish between these mechanisms. In contrast to the specific inhibition of carbachol-induced phosphatidylinositol turnover by PMSF, PMSF has a transient inhibitory effect on both carbachol-induced and potassium-induced contractions. The non-selective effect of PMSF on contraction suggests that this effect does not stem from its inhibition of phosphatidylinositol degradation. In the presence of Li+, PMSF (2 mM) has an inhibitory effect of only 15%-19% on carbachol-induced inositol phosphate accumulation, suggesting that the inhibition of phosphatidylinositol turnover by PMSF is not due to its inhibition of phosphatidylinositol phosphodiesterase, but rather to its inhibition of one or more steps following the degradation of phosphatidylinositol. [3] Benzyl sulfonyl fluoride (PMSF) has been shown to inhibit the binding of ethanolamine phosphate to glycosyl phosphatidylinositol (GPI) intermediate in Trypanosoma brevicornu (Masterson, WJ, and Ferguson, MAJ (1991) EMBO J. 10, 2041-2045). This study demonstrates that the Man3-GlcN-PI intermediate accumulated in the presence of PMSF can undergo fatty acid remodeling, suggesting that fatty acid remodeling enzymes are not specifically targeted at GPI intermediates containing ethanolamine phosphate. We also found that PMSF inhibited the acylation of inositol residues in GPI intermediates in blood trypanosomes. Pulse-tracking experiments showed that glycolipid C (ethanolamine-PO4-Man3-GlcN-(acyl)PI) is not a necessary precursor to glycolipid A (ethanolamine-PO4-Man3-GlcN-PI), and glycolipid C can be converted to glycolipid A. These data suggest that glycolipid C is the final product of the GPI biosynthetic pathway and exists in dynamic equilibrium with glycolipid A. The inhibitory effect of PMSF on ethanolamine phosphate addition and inositol acylation in the procyclic trypanosome was also observed in trypanosomes, but not in mammalian HeLa cells. These results indicate differences between the relevant parasitic enzymes and mammalian enzymes. [4]

---

Anandamide is a putative endocannabinoid ligand with pharmacological effects similar to those of Δ9-tetrahydrocannabinol (THC), the main psychoactive component of cannabis. Extensive evidence suggests that the enzyme inhibitor phenylmethylsulfonyl fluoride (PMSF) can alter the effects of arachidonic acid ethanolamine in vitro by blocking its metabolism. Therefore, this study was conducted in mice to determine whether PMSF can produce cannabinoid effects by altering endocannabinoid ethanolamine levels and whether PMSF can enhance the effects of exocannabinoid ethanolamine. Mice injected intraperitoneally with PMSF exhibited cannabinoid effects, including analgesia, hypothermia, and reduced activity, with ED50 values of 86, 224, and 206 mg/kg, respectively. Spontaneous activity decreased in mice at doses exceeding 100 mg/kg. However, these effects were not blocked by the cannabinoid antagonist SR 141716A. On the other hand, pre-administration of an ineffective dose of PMSF (30 mg/kg) enhanced the effects of arachidonic acid ethanolamine (anandamide) on tail-flicking response (analgesia), spontaneous activity, and mobility by 5, 10, and 8 times, respectively. PMSF did not affect the hypothermic effect of arachidonic acid ethanolamine. In general, these findings of PMSF highlight the importance of metabolism in the action of arachidonic acid ethanolamine. Whether the metabolites of arachidonic acid ethanolamine contribute to its pharmacological activity remains to be determined. [5]

---

The serine/cysteine protease inhibitor benzyl sulfonyl fluoride (PMSF) has been used to promote and protect hens from the neuropathological events of organophosphate-induced delayed neuropathy (OPIDN) (Veronesi and Padilla, 1985; Pope and Padilla, 1990; Lotti et al., 1991; Pope et al., 1993; Randall et al., 1997). This study, for the first time, expanded upon this work by utilizing high-resolution microscopy provided by epoxy resin embedding and ultrathin sectioning techniques to assess the neuropathological manifestations of promoting and protective effects and their correlation with associated clinical changes. To assess the dose-related effects of OPIDN, adult hens were injected with a single dose of phenylsalicylic acid phosphate (PSP) at doses of 0.5, 1.0, or 2.5 mg/kg. PMSF (90 mg/kg) was administered 4 hours after PSP administration (promoting effect) or 12 hours before administration (protective effect). Clinical symptoms and pathological changes in the digastric nerve, which has a unique sensitivity to OPIDN (El-Fawal et al., 1988), were monitored. PSP alone (2.5 mg/kg) induced severe OPIDN (final clinical score 7.5 ± 1.0 [0-8 scale]; neuropathological score 2.7 ± 0.3 [0-4 scale, based on myelin fiber degeneration]). Administration of PMSF 12 hours prior to PSP administration completely protected OPIDN (both clinical and neuropathological scores were 0; p<0.0001 compared to PSP alone). No signs or lesions of OPIDN were observed after PSP alone (0.5 mg/kg), but administration of PMSF 4 hours after PSP administration enhanced its neurotoxicity (all hens had a clinical score of 4.0, and the mean neuropathological score was 3.5 ± 0.3; p<0.0001 compared to PSP alone). Although quantitative differences were observed, no qualitative differences were found in the nerve tissue of hens with OPIDN under light and electron microscopy. At euthanasia, a statistically significant linear relationship was observed between clinical and neuropathological scores on the last day of observation (r² = 0.76, p<0.0001). This study indicates that the degree of peripheral nerve myelin fiber degeneration is associated with clinical deficiencies in the enhancement and protection of OPIDN induced by PMSF. [6] PMSF is a synthetic serine protease inhibitor that is irreversible at high concentrations and reversible at low concentrations. It is widely used as a research tool to study the role of serine proteases in enzymatic reactions, cell signaling, and pathological processes such as inflammation and tissue damage. [3,4] Early studies ([1],[2]) focused on the ability of PMSF to inhibit plasma and tissue serine proteases such as plasmin and carboxylesterase, laying the foundation for its application in coagulation and metabolic pathway studies. [1,2] In cardiovascular studies, PMSF is used to inhibit calpain (a serine protease-associated enzyme) and reduce cardiomyocyte damage caused by OPIDN. Oxidative stress [4] - In neuroscience, PMSF has shown neuroprotective effects in models of cerebral ischemia and excitotoxicity by inhibiting serine protease-mediated neuronal death, supporting its use as a tool for studying the mechanisms of neurodegenerative diseases [5,6]. - PMSF is unstable in aqueous solution (half-life of approximately 30 minutes at neutral pH), therefore it needs to be freshly prepared before experiments [3].

C7H7FO2S

174.19

174.015

C, 48.27; H, 4.05; F, 10.91; O, 18.37; S, 18.41

329-98-6

4784

White to off-white solid powder

1.3±0.1 g/cm3

285.7±19.0 °C at 760 mmHg

92-95 °C

126.6±21.5 °C

0.0±0.6 mmHg at 25°C

1.522

2.33

0

3

2

11

199

0

O=S(CC1=CC=CC=C1)(F)=O

YBYRMVIVWMBXKQ-UHFFFAOYSA-N

InChI=1S/C7H7FO2S/c8-11(9,10)6-7-4-2-1-3-5-7/h1-5H,6H2

phenylmethanesulfonyl fluoride

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)

Isopropanol: ~60 mg/mL (~344.5 mM)

DMSO: ~35 mg/mL (~200.9 mM)

Water: <1 mg/mL

Ethanol: ~15 mg/mL (~86.1 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (11.48 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2 mg/mL (11.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 2 mg/mL (11.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 20 mg/mL (114.82 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

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