Bruno Mariela A, Trejo Sebastián A, Avilés Xavier F, Caffini Néstor O, López Laura M I
Laboratorio de Investigación de Proteínas Vegetales (LIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, C.C. 711, La Plata B1900AVW, Argentina.
Protein J. 2006 Apr;25(3):224-31. doi: 10.1007/s10930-006-9005-8.
From unripe fruits of Bromelia hieronymi Mez (Bromeliaceae), a partially purified protease preparation was obtained by acetone fractionation of the crude extract. Purification was achieved by anionic exchange chromatography (FPLC) on Q-Sepharose HP followed by cationic exchange chromatography (SP-Sepharose HP). Homogeneity of the new enzyme, named hieronymain II, was confirmed by SDS-PAGE and mass spectroscopy (MALDI-TOF-TOF). The molecular mass of was 23,411 Da, and maximum proteolytic activity (more than 90% of maximum activity) was achieved at pH 7.5-9.0 on casein and at pH 7.30-8.3 on Z-Phe-Arg-p-nitroanilide. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine. The N-terminal sequence of hieronymain II (AVPQSIDWRVYGAV) was compared with those of 12 plant cysteine proteases which showed more than 70% of identity. Kinetic enzymatic assays were made on Z-Phe-Arg-p-nitroanilide (Km = 0.72mM, kcat = 1.82 seg(-1) , kcat/ Km = 2.54seg(-1) mM(-l)). No detectable activity could be found on PFLNA or Z-Arg-Arg-p-nitroanilide.
从凤梨科植物希氏凤梨（Bromelia hieronymi Mez）的未成熟果实中，通过对粗提物进行丙酮分级分离获得了一种部分纯化的蛋白酶制剂。通过在Q-Sepharose HP上进行阴离子交换色谱（FPLC），随后在SP-Sepharose HP上进行阳离子交换色谱来实现纯化。通过SDS-PAGE和质谱（MALDI-TOF-TOF）确认了名为希氏蛋白酶II（hieronymain II）的新酶的同质性。其分子量为23,411 Da，在pH 7.5 - 9.0时对酪蛋白具有最大蛋白水解活性（超过最大活性的90%），在pH 7.30 - 8.3时对Z-Phe-Arg-p-硝基苯胺具有最大活性。该酶被E-64和碘乙酸完全抑制，并通过添加半胱氨酸而被激活。将希氏蛋白酶II的N端序列（AVPQSIDWRVYGAV）与12种植物半胱氨酸蛋白酶的序列进行了比较，这些序列显示出超过70%的同一性。对Z-Phe-Arg-p-硝基苯胺进行了酶动力学测定（Km = 0.72mM，kcat = 1.82 s⁻¹，kcat/Km = 2.54 s⁻¹ mM⁻¹）。在PFLNA或Z-Arg-Arg-p-硝基苯胺上未检测到可检测的活性。
