The pepsinogens and pepsins of cobra, mole snake and puff adder have been studied. The pepsinogens of all three species fall into two distinct groups, here designated PI and PII. At least the latter group, in all cases, shows substantial microheterogeneity. Physicochemical studies suggest that the cobra and puff adder PII groups are more similar to each other than either is to the mole snake PII group. Kinetic studies indicate that, in the cobra and mole snake, the PI and PII pepsins differ in their Arrhenius activation energies. Such difference is smaller, or absent, in the case of the puff adder PI and PII pepsins. These characteristics of the pepsins are assessed in the context of the differences between the oral secretions of the three species studied. The suggestion is advanced that the puff adder's strongly proteolytic venom has influenced certain properties of its gastric proteases.

Using proteolytic probes, the pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was analyzed for its native topography in the cytoplasmic membrane. Before analyses could be performed, the isolation of transhydrogenase-enriched ISO (inside-out) cytoplasmic membrane vesicles was accomplished by modification of the procedure followed by Clarke (Clarke, D. M. and Bragg, P. D. (1985) Eur. J. Biochem. 149, 517-523) in purifying the enzyme from overexpressing E.coli JM83pDC21 cells. Two major changes were made. One was that the solubilization of the bacterial membrane and subsequent purification steps were omitted. The other was the separation of outer membranes from the cytoplasmic membrane preparation by sucrose gradient density centrifugation. This was essential owing to the contaminating presence of a 30 kD protein in the outer membrane of the original preparation. Transhydrogenase-enriched RSO (right-side-out) membrane vesicles were isolated by a different procedure using lysozyme-mediated breakage of E.coli spheroplasts and subsequent vesicular reformation. To identify possible transhydrogenase fragments arising from proteolytic cleavage, anti-E.coli transhydrogenase polyclonal antibodies were generated in rabbits. Two sets of polyclonal antibodies were produced. One set cross-reacted with both the α (52 kD) and β (48 kD) subunits of the transhydrogenase. The other reacted with the α subunit only. Trypsin and proteinase K were the main proteolytic probes used against both ISO and RSO cytoplasmic membrane vesicles, although chymotrypsin was also used in preliminary experiments with ISO membrane vesicles. Identification of fragments resulting from proteolytic cleavage of the enzyme was obtained using anti-transhydrogenase antibodies and by N-terminal sequencing and/or C-terminal sequencing. In some of these experiments, isolation of the proteolytic fragments was necessary prior to analysis. This was done using a number of different methods. The particular methods applied, which included column chromatography strategies and elution procedures from SDS-Polyacrylamide gels, depended on the type of analysis carried out. The analyses indicated that the α subunit has at least a 41 kD sequence extending from its N-terminus which is exposed to the cytoplasmic side of the membrane. This sequence may contain an active site of the enzyme. This is suggested by the binding of this fragment to a NAD-affinity column. The membrane-imbedded region of the α subunit anchoring the 41 kD region predicted by hydropathy plotting (Clarke, D. M., Loo, Tip W., Gilliam, S. and Bragg, P. D. (1986), Eur. J. Biochem. 158, 647-653) could not be detected by our methods. Susceptible tryptic cleavage sites along the 41 kD region were identified by partial proteolysis and may reflect areas in the subunit's tertiary or quaternary structure that are exposed to the surrounding medium. Major cleavage sites were at arg₁₅, Iys₂₂₇, Iys₂₆₄, arg₂₆₈, Iys₂₇₅, arg₃₅₅, and arg₃₆₁. There do not appear to be significant portions of the subunit protruding into the periplasm as neither trypsin nor proteinase K had any effect on the subunit in RSO-oriented membrane vesicles. Proteinase K experiments with ISO and RSO membrane vesicles suggest that a 20 kD portion of the β subunit is protected from cleavage and is imbedded in the membrane. The identity of this fragment could not be confirmed. Hydropathy analysis of the transhydrogenase gene-derived amino acid sequence (Clarke, D. M., Loo, Tip W., Gilliam, S. and Bragg, P. D. (1986), Eur. J. Biochem. 158, 647-653) suggests that this could be a sequence extending from the N-terminus of the β subunit. This is a hydrophobic sequence containing 7 possible transmembranous helices and having a theoretical molecular weight in the range of 20 kD. The proteinase K results also indicate that the rest of the β subunit is exposed to the cytoplasmic side of themembrane rather than the periplasmic side. The results obtained here are consistent with hydropathy predictions made with regard to this subunit. In addition, two different experiments indicate that an α-α subunit interaction may be present in the oligomeric structure of the membrane-bound enzyme (Hou, C, Potier, M. and Bragg, P. D. (1990), Biochim. Biophys. Acta 1018, 61-66). Substrates of the enzyme did not appear to affect the transhydrogenase's general conformation upon binding as detected by experiments using partial tryptic proteolysis. Partial trypsinolysis also revealed that selective detergent extraction of transhydrogenase-enriched ISO vesicles with Triton X-100 and sodium cholate did not affect the overall conformation of the membrane-bound enzyme despite greatly reducing the enzymatic activity. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

In the first chapter, sodium tetrathionate (TT), a sulfhydryl blocking agent, is assessed for its ability to protect the proteolytic activity (PA) of papaya latex during air, sun or vacuum drying, and of crude papain during storage. By means of Taguchi's L₂₇ (3¹³) fractional factorial design, it was found that the addition of 1% TT significantly increased the retention of PA of papaya latex when it was air dried at a temperature of 55°C. This protection of PA was found to be 23% higher than the one given by the addition of 1% sodium metabisulfite, the compound commonly used in the commercial processing of papaya latex. When drying was carried out either under 27 inches vacuum at 50°C or in the sun, the protective effect of TT on the PA was not significantly different from that of metabisulfite. The PA of crude papain during storage at room temperature was also protected by TT. A loss of 20% of the original PA occurred over a period of 13 wk when crude papain contained 1% TT, compared to a loss of 45% when the crude enzyme preparation contained 1% metabisulfite. In the same chapter five different oxidants for synthesis of TT from thiosulfate are compared, namely: iodine, hydrogen peroxide, ferric chloride, cupric sulfate and sodium vanadate. The results indicated that hydrogen peroxide or sodium vanadate were not only effective in the oxidation but also much less expensive than iodine, which is the most popular oxidant for the synthesis of TT. The results obtained in this chapter warrant the use of TT in the commercial production of commercial papain to prevent the destruction of the enzymes during harvesting, storage, transportation and processing. In the second chapter, chemical modification of pure papain by TT is discussed. Optimization techniques were applied for improving the precision of two methods used in this study: circular dichroism (CD) and proteolytic activity determination. Simplex optimization significantly improved repeatability and signal to noise ratio of the CD scan of papain. A new optimization approach, which was a combination of a central composite rotatable design and simplex optimization, was successfully applied to achieve maximum precision for the proteolytic activity assay of papain using casein as a substrate. This approach may also be applied to other analytical methods to improve the reliability of the experimental data. Influential factors in the inactivation of PA of papain by using TT and reactivation of the inactivated papain by cysteine were carried out using two Taguchi's L₁₆ (2¹⁵) fractional factorial designs. The results indicated that when inactivation was carried out at pH 6.8, with a reaction time of 5 min at 22°C, and a molar ratio of TT to papain of 10, the inactivation reaction was highly reversible upon addition of 20 mM cysteine. Although some interactions of the factors were significant, 70% reactivation was achieved in most cases. Analysis of UV absorbance, near-UV and far-UV CD spectra indicated that there were no major changes in the spectra in papain upon the chemical modification of the enzyme with TT. Secondary structure computed from far-UV CD spectra also demonstrated no significant changes upon this modification. Sulfhydryl data and pH-fluorescence profiles of the modified papain support the hypothesis that reversible blocking by TT results from binding with the single reactive cysteine residue present in papain. Quenching of the intrinsic fluorescence of papain when the modification was carried out using high molar ratios of TT to papain was suggestive of modification of tryptophan residues in the enzyme during the oxidation reaction with TT. Precipitation or insolubilization of pure papain, and of the proteins of papaya latex and commercial papain was observed upon the chemical modification with TT under certain conditions. Addition of β-mercaptoethanol and TT at levels of 100 mM and 50 mM, respectively, precipitated 90% of pure papain. Solubility studies together with electrophoretic analysis of the precipitated papain suggested formation of insoluble aggregates due to the insoluble aggregation as a result of inter-molecular disulfide bonds formation. TT was found to be a competitive inhibitor of both reversible and irreversible inhibition of the enzyme action, when carbobenzoxyglycine p-nitrophenyl ester was used as a substrate. The second order inactivation constant in the absence of substrate was computed to be 16,919 M⁻¹sec⁻¹, indicating that the reaction had a high rate. / Land and Food Systems, Faculty of / Graduate

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: a) Characterization of traditional methodologies and testing methods used to purify and quantify trypsin and α-chymotrypsin b) Re-engineering / development of a new method for purifying trypsin and α-chymotrypsin that delivered higher product yields and improved control exercised over the process by investigating: i. Extraction methods ii. Centrifugation iii. Ultrafiltration iv. Chymotrypsinogen and trypsin crystallization v. Column chromatography vi. Investigation into different raw material sources for pancreatic enzyme production c) Development of kinetic and ELISA testing methodologies for in-process QC analysis. / AFRIKAANSE OPSOMMING: Hierdie Studie beskryf: a) Karakterisering van die ou prosessering metodes en toets metodes wat gebruik word om Tripsien en Alpha-chimotripsien te suiwer en te kwantifiseer. b) Herontwerp / ontwikkeling van 'n nuwe metode vir die suiwering Tripsien en Chimotripsien wat „n hoër opbrengs lewer en meer kontrole oor die proses uit oefen deur ondersoek in te stel na: i. Ekstraksie- metodes ii. Sentrifugering iii. Ultrafiltrasie iv. Chymotripsienogeen - en tripsien kristallisasie v. Kolom chromatografie vi. Ondersoek na verskillende rou materiaal bronne vir die produksie van pankreas ensieme. c) Die ontwikkeling van kinetiese- en ELISA toets metodes vir die in-proses kwaliteitkontrole.

Kinetic properties of the two proteases, causing textural degradation of Pacific whiting (Merluccius productus) during heating, were compared and characterized with the synthetic substrate, Z-Phe-Arg-NMec. Pacific whiting P-I and P-II showed the highest specificity on Z-Phe-Arg-NMec, specific substrate for cathepsin L. The K [subscript m] of preactivated P-I and P-II were 62.98 and 76.02 (μM), and k [subscript cat], 2.38 and 1.34 (s⁻¹) against Z-Phe-Arg-NMec at pH 7.0 and 30°C, respectively. Optimum pH stability for preactivated P-I and P-II is between 4.5 and 5.5. Both enzymes showed similar pH-induced preactivation profiles at 30°C. The maximal activity for both enzymes was obtained by preactivating the enzyme at a range of pH 5.5 to 7.5. The highest activation rate for both enzymes was determined at pH 7.5. At pH 5.5, the rate to reach the maximal activity was the slowest, but the activity was stable up to 1 hr. P-I and P-II shared similar temperature profiles at pH 5.5 and pH 7.0 studied. Optimum temperatures at pH 5.5 and 7.0 for both proteases on the same substrate were 55°C. Significant thermal inactivation for both enzymes was shown at 75°C. Preactivated P-I and P-II displayed a similar first order thermal inactivation profile at pH 7.0. At 30 and 90°C, half lives, t [subscript 1/2], for Pacific whiting P-I were 49.50 and 0.20 min and for P-II, 32.54 and 0.18 min, respectively. The rate constant of inactivation for both proteases increased about 200-fold between two limits, 30 and 90°C. Half lives at 55°C, optimum temperature, for P-I and P-II were also determined to be 5.29 and 6.75 min. The increase in thermal inactivation rate constants independent of substrates corresponded to an activation energy for heat denaturation of 21.18 kcal/mol for P-I and 19.97 kcal/mol for P-II by Arrhenius plot. These similar kinetic properties, i.e., kinetic parameters, pH profile and thermal inactivation rate constant, suggested that Pacific whiting P-I and P-II are the same enzyme. / Graduation date: 1994

Wretlind, Heden, and Wadstrom found ammonium sulfate to be inhibitory for the formation of extracellular protease in Aeromonas hydrophila grown in Brain Heart Infusion medium. They demonstrated by manipulating the iron and zinc content within their medium that it is possible to differentially affect the accumulation of hemolysin and protease by A. hydrophila grown in batch culture. Further manipulation of the composition of this medium was done in the present study to determine the effect of other components on the production of protease. The purpose of this study was to determine the factors affecting the level of A. hydrophila protease produced in a chemically defined medium.

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal region of TIMP and the C-D B-strand connector which occupy the primed (right side of the active site) and unprimed (left side) regions of the active site. Substitutions for Thr2 of N-TIMP- 1 strongly influence MMP selectivity. In this study we found that Arg and Gly, which generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the NTIMP-1 mutant with AB loop of TIMP-2, it produced a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and MMP-9, respectively. The Gly mutant has a Ki of 2.1 nM for MMP-9 and > 40 uM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily. In collaboration with Dr. Yingnan Zhang at Genentech, we have developed a protocol for the phage display of full-length human TIMP-2 to identify high-affinity selective inhibitors of human MMP-1, a protease that plays a role in cleaving extracellular matrix (ECM) components, connective tissue remodeling during development, angiogenesis, and apoptosis. We have generated a library containing 2x1010 variants of TIMP-2 randomized at residues 2-6 (L1), at residues 34-40 (L2) and 67-70 (L3). / The L1 library yielded a positive signal for MMP-1 binding. Clones from the L1 library, designated TM1, TM8, TM13, and TM14, were isolated after 5 rounds of selection on immobilized MMP-1 and MMP-3 and found to show a greater selectivity for MMP-1 relative to MMP-3. TM8, which has Ser2 to Asp and Ser4 to Ala substitutions, showed the greatest apparent selectivity of 10-fold toward MMP-1 compared to MMP-3. The various mutations identified by phage display were introduced into recombinant Nterminal TIMP-2 and the variants characterized as inhibitors of an array of MMP catalytic domains. The TM8-based mutant showed pronounced selectivity (> 1000-fold for MMP-1 vs. MMP-3) and may be a step towards the generation of MMP-1-specific inhibitors. Molecular modeling was used to rationalize the structural basis of MMP selectivity in the mutants. / by Harinathachari Bahudhanapati. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

The 23 matrix metalloproteinases (MMPs) in humans catalyze the turnover of all protein components of the extracellular matrix (ECM) and have important roles in tissue remodeling, wound healing, embryo implantation, cell migration and shedding of cell surface proteins. Excess MMP activities are associated with many diseases including arthritis, heart disease and cancer. The activities of MMPs are regulated by a family of four protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), that are endogenous inhibitors of matrix metalloproteinases (MMPs), ADAMs (A Disintegrin And Metalloproteinase) and ADAMTS (disintegrin-metalloproteinase with thrombospmdin motifs) .... The balance between TIMPs and active metzinicins is very important and imbalances are linked to human diseases such as arthritis, cancer, and atherosclerosis. The engineering of TIMPs to produce specific inhibitors of individual MPs could provide new therapeutic principles for disease treatment, but this requires a detailed understanding of the biophysical and structural basis of the interactions of TIMPs and MMPs and ADAMs. / by Wu Ying. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Matrix metalloproteinases (MMPs) constitute the major class of enzymes capable of degrading all protein components of extracellular matrix (ECM) and have important roles in normal physiologic processes of maintaining tissue integrity and remodeling. However, excess MMP activities are associated with many diseases including rheumatoid arthritis and osteoarthritis, cardiomyopathy, and macular degeneration. The activity of MMPs is regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) which are avid broad-spectrum inhibitors of numerous human matrixins (MMPs and ADAMs). Uncontrolled matrix degradation occurs when the balance between TIMPs and MMPs is disrupted, resulting in serious diseases such as cancer, arthritis and chronic tissue ulcers. Thus, the engineering of TIMPs to produce highly selective and efficacious inhibitors of individual MMPs may be utilized for future treatment of diseases. Such engineering requires detailed analysis for the structural and biophysical information of MMP-TIMP interaction. Changes in the dynamics of proteins and solvent that accompany their associations with different binding partners, influence the specificity of binding through entropic effects. From the current studies it appears that the interactions of the inhibitory domains of TIMPs-1 and -2 (N-TIMPs) with MT1-MMP are driven by entropy increases that are partitioned between solvent and conformational entropy (ΔSsolv and ΔSconf), and a large conformational entropy penalty is responsible for the weak inhibition of MT1-MMP by NT1.We investigated how mutations that modify N-TIMP selectivity affect the thermodynamics of interactions with MMP1, MMP3 and MT1-MMP. The weak inhibition of MT1-MMP by N-TIMP-1 is enhanced by mutation of threonine 98, on the edge of the binding ridge, to leucine. This mutation increases the large ΔSconf cost for binding to MT1-MMP but this is offset by a greater increase in ΔSsolv. In contrast, this mutation enhances binding to MMP3 by increasing ΔSconf for the interaction. ΔSsolv and ΔSconf show mutual compensation for all interactions, with characteristic ranges for each MMP. Distinct electrostatic and dynamic features of MMPs are key factors in their selective inhibition. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

by Lau King-Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 119-142). / ACKNOWLEDGMENTS --- p.I / ABSTRACT --- p.II / ABBREVIATIONS --- p.IV / TABLE OF CONTENTS --- p.V / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- PURIFICATION OF ADH-I & ADH-C --- p.25 / Chapter CHAPTER 3 --- "PURIFICATION & IDENTIFICATION OF ""ADH-ACTIVATING"" PROTEASE" --- p.60 / Chapter CHAPTER 4 --- ACTIVATION OF ADH-I BY COMMERCIAL PROTEASE & BY ACETIMIDYLATION --- p.90 / Chapter CHAPTER 5 --- CONCLUSION --- p.114 / REFERENCES --- p.118