PURIFICATION AND CHARACTERIZATION OF A TRYPSIN INHIBITOR FROM PUTRANJIVA ROXBURGHII

Abstract

Protein proteinase inhibitors have been found in many plant tissues especially in legume seeds and other storage organ, numerous animal tissue and fluids and in microorganism. Serine proteinase inhibitors are widely distributed in nature and have been isolated from many sources including animals, plants and microorganisms. Plant seeds are rich source of inhibitors. Many plant serine proteinase inhibitors have been purified and characterized particularly from the seeds ofLeguminosae, Cucurbitaceae, Solanaceae and Gramineae family. There are not many reports of purification and characterization of these inhibitors from other plant families. Other families where an inhibitor has been purified and characterized include Rutaceae and Euphorbiaceae. Putranjiva roxburghii belonging to Euphorbiaceae family is an ornamental tree of tropical India, known as child life tree. Deseeded fruits are used against cough, cold and sprue. Rosaries of hard stones are used for protecting children from infections. The seed kernel on steam distillation yield 0.5% of a sharp-smelling essential oil of the mustard oil type. The oil contains isopropyl and 2-butyl isothiocyanates as the main constituents and 2-methyl-butyl isothiocyanate as aminor component. Roxburghonic keto acid and some flavonoids, terpenoids and triterpines has been purified and characterized from the leaf and trunk bark of this plant. To date, no protein has been characterized from this plant. This work describes the purification and characterization of a highly stable and potent trypsin inhibitor from the seeds of Putranjiva roxburghii. Chapter 1 reviews the literature in the area of plant proteinase inhibitors particularly serine proteinase inhibitors. Chapter 2 describes the purification of a Kunitz-type trypsin inhibitor from the seeds of Putranjiva roxburghii. The Putranjiva roxburghii trypsin inhibitor (PRTI) was purified to homogeneity in three steps by acid precipitation, CM-sepharose cation exchange and DEAEsepharose anion exchange chromatography. In acid precipitation step, low molecular mass proteins were precipitated along with some other proteins. The trypsin inhibitory activity was retained in supernatant. In second step on CM-sepharose column, all the low molecular mass proteins were bound to the column while trypsin inhibitory activity was found in flow through. In last step, protein with trypsin inhibitory activity was bound to a DEAE-sepharose column. After washing the column extensively, bound proteins were eluted with step gradient of NaCl. The fractions with trypsin inhibitory activity were eluted at 50 and 100 mM NaCl. The purity of the protein in above fractions was analyzed by SDS-PAGE. The fraction eluted at 100 mM NaCl showed single band on SDS-PAGE. The protein was further subjected to size exclusion chromatography column on HPLC. The purified protein showed single band on SDS-PAGE. The SDS-PAGE analysis under both reducing and non-reducing conditions showed that PRTI is a single polypeptide chain with a molecular mass of approximately 34 kDa. Chapter 3 describes the biochemical characterization of Putranjiva roxburghii trypsin inhibitor. Amino acid sequence analysis was performed by Edman degradation and mass spectrometry studies. In N-terminal sequencing of PRTI, first 10 residues from the N-terminal were obtained. The sequence determined was Arg-Pro-Pro-Gln-Ala-Gly-Tyr-Ile-Gly-Val. The N-terminal sequence of PRTI showed no similarities with any of the known trypsin inhibitors. In partial internal sequencing, sequences of four peptides were obtained. In separate experiments, one peptide (Peptide 1) was obtained from LC-MS/MS and three peptides from MALDI-TOF/TOF studies. The peptide sequenced by LC-MS/MS analysis showed identity to winged bean chymotrypsin inhibitor-3. The inhibitory activity of PRTI against trypsin and chymotrypsin were determined by measuring the hydrolytic activity toward BAPNA and BTEE respectively. The protein completely inhibited trypsin at a molar ratio of 1:1 but did not show any significant inhibition against a-chymotrypsin. The analysis ofDixon plot showed that the PRTI is a competitive inhibitor where two lines corresponding to each substrate intersect above the x-axis, a characteristic of competitive inhibition. The dissociation constant (Ki) value was found to be 1.4 x 10~n M. In stability studies, effect oftemperature, pH and DTT was examined on inhibitory activity of PRTI. In thermal stability studies, trypsin inhibitory activity ofPRTI was completely retained up to 70 °C. Above 70 °C, there was a slight decrease in the inhibitory activity with PRTI retaining almost 85% inhibitory activity up to 80 °C. The inhibitory activity ofPRTI fell sharply above 80 °C with a loss ofalmost 80% inhibitory activity at 90 °C. In pH stability studies, PRTI was highly stable under conditions ranging from highly acidic to highly alkaline. PRTI showed maximum inhibition atpH 8.0 and maintained over 95% of its inhibitory activity through apH gradient of 2-12. In presence of DTT, PRTI was found completely stable with no loss in inhibitory activity when incubated for 1h up to 100 mM DTT. Only a slight decrease of5% in inhibitory activity was observed when PRTI was incubated for 2hat 100 mM DTT. Purified PRTI proteins were used for proteolysis studies with different proteases. Purified protein was incubated with five different proteinases, namely trypsin, chymotrypsin, papain, pepsin and proteinase K, separately using aprotease/PRTI molar ratio of 1:50 for different time periods ranging from 30 min to 24 hat room temperature. All samples were analyzed on a 15% SDS-PAGE. PRTI is very stable against trypsin, chymotrypsin and pepsin and the results obtained did not show any cleavage. Although, PRTI is a serine proteinase inhibitor but proteolytically it is very stable against aspartate proteinases like pepsin. PRTI is partially in cleaved by papain and completely cleaved by proteinase k enzyme. Time dependant proteolytic cleavage studies were also performed to determine any domain structure of PRTI but there was no such difference observed in the cleavage pattern. PRTI retained almost 90% inhibitory activity after one year storage at -20 °C. Chapter 4 describes the biophysical characterization of PRTI by circular dichroism and fluorescence studies. Far-UV CD spectroscopy studies (240 -200 nm wavelength range) were carried out to analyze the secondary structure and conformational stability of PRTI at different temperatures from 20 to 100 °C. Analysis ofCD spectra of native PRTI showed that it is an a, P protein with negative peaks at around 217 nm and 208 nm. Although, negative ellipticity was present but no clear negative peak characteristic of a-helical structures was observed at 222 nm. These results strongly suggest that PRTI is a, P protein rather than predominantly P protein. CD studies at increasing temperature demonstrated the thermo stability of PRTI structure. The PRTI retained the back bone protein folding with no significant change in CD spectra up to 70 °C. A significant loss in ellipticity was observed at and above 90 °C. This correlates well with the results of inhibitory activity where 15% loss was observed at 80 °C and 80% at 90 °C. The inhibitory activity and CD studies at increasing temperatures showed that transition midpoint for PRTI lies close to 88 °C. Fluorescence spectroscopy experiments were performed in different physicochemical conditions to monitor the extent of changes in native structure of PRTI and relate them to the inhibitory activity. Extrinsic (ANS) and intrinsic (tryptophan) fluorescence monitoring studies of conformational stability exhibited that PRTI gradually unfolds as the concentration of GuHCl and Urea increases and above 8M, completely unfolded molten globule structure present. PRTI lost its native conformation after incubation in the range of 3-5% SDS and 200-1000 mM HCl concentration. Fluorescence emission spectra analysis significantly IV correlates the structure-activity relationship when studied as a function of DTT, pH and temperature denaturation. PRTI structure and inhibitory activity was retained up to 100 mM DTT, 80 °C temperature and in highly alkaline and acidic pH ranging from 2.0-12. In comparison to alkaline pH, PRTI exhibited little higher unfolding at acidic pH.