PMSF is an irreversible serine protease inhibitor that is often added to the lysis buffer before cell or tissue lysis to prevent protein degradation[1,2]. PMSF can effectively inhibit chymotrypsin, trypsin, thrombin, etc. The principle of action of PMSF is that it can specifically recognize and sulfonate the serine residue active site that determines enzyme activity. PMSF is also an inhibitor of fatty acid amide hydrolase (FAAH), cysteine proteases (such as papain, the inhibitory activity of which is reversed by DTT), and acetylcholinesterase of mammalian origin[3]. The inhibitory effect of PMSF on papain can be reversed by DTT (Dithiothreitol).
The deactivation of PMSF increases with increasing pH and temperature. The half-lives of the inhibitors are approximately 110, 55, and 35 minutes at 25°C and pH 7.0, 7.5, and 8.0, respectively. At pH 8, 100μM PMSF is almost completely inactivated within 1 hour at 25°C or 22 hours at 4°C. PMSF stock solutions prepared using 100% isopropyl alcohol are stable for months or longer at 25°C. Within 1 week at 25°C and pH 7.0, chymotrypsin inhibited by PMSF did not reactivate[4].
In animal experiments, PMSF had significant analgesic effects at doses of 120 and 300 mg/kg. Sub-analgesic doses (60 mg/kg, i.p.) of PMSF can reduce tolerance resulting from acute and chronic administration of opioids[3].
PMSF can be used alone or often mixed with other protease inhibitors such as aprotinin A or phosphatase inhibitors such as sodium orthovanadate.
PMSF is poorly soluble in water and has poor stability in aqueous solutions. It is recommended to add it to lysis buffer or other aqueous solutions before use. PMSF powder can be dissolved in methanol, ethanol or isopropyl alcohol to prepare a storage solution. The recommended concentration is 100-200mM. The effective working concentration of PMSF is 0.1-1mM.
PMSF (苯甲基磺酰氟)是一种不可逆的丝氨酸蛋白酶抑制剂，常在细胞或组织裂解之前加入裂解液中以预防蛋白的降解[1,2]。PMSF能够有效抑制胰凝乳蛋白酶、胰蛋白酶、凝血酶等。PMSF的作用原理是能够特异性识别并磺化决定酶活性的丝氨酸残基活性位点。PMSF也是脂肪酸酰胺水解酶 (FAAH) 、半胱氨酸蛋白酶(如木瓜蛋白酶，DTT逆转其抑制活性)和哺乳动物来源的乙酰胆碱酯酶的抑制剂[3]。PMSF对木瓜蛋白酶的抑制效果可被DTT（Dithiothreitol，二硫苏糖醇）逆转。
PMSF的失活随着pH值和温度的升高而增加。在25°C和pH 7.0、7.5和8.0时，抑制剂的半衰期分别约为110、55和35分钟。在pH 8时，100μM PMSF在25°C下1小时内或在4°C下22小时几乎完全失活。使用100%异丙醇制备的PMSF储备溶液在25°C下可稳定保存数月甚至更长。在25°C、pH 7.0的条件下1周内，PMSF抑制的胰凝乳蛋白酶没有发生再激活[4]。
在动物实验中，PMSF在剂量为120和300mg/kg时具有显着的镇痛作用。亚镇痛剂量 (60 mg/kg, i.p.) 的PMSF可以降低阿片类药物的急性和慢性给药导致的耐受性[3]。
PMSF可以单独使用，也常与其他蛋白酶抑制剂如胃抑肽酶A或者磷酸酶抑制剂如正钒酸钠混合使用。
PMSF难溶于水，且在水溶液中的稳定性很差，建议在使用前加入裂解液或其他水溶液中。PMSF粉末可用甲醇，乙醇或者异丙醇溶解配置储存液，推荐浓度100-200 mM。PMSF的有效工作浓度为0.1-1 mM。