A proteolytic activity has been shown to be associated with the heat shock protein 70 (hsp70). In order to study this, I have constructed RNA transcribing vectors with the coding sequences of the D. melanogaster (pBUG7) and the human (pMAN70) genes coding hsp70, and with an internal deletion (pBUG301) in D. melanogaster. Proteins from 37 kDa to 70 kDa were translated in a rabbit reticulocyte lysate in the presence of 35S-methionine from RNA synthesized in vitro off the full length templates (pBUG7, and pMAN70), or altered templates. Restriction digestion of pBUG7 with BamH I and Nar I yields templates that produce carboxy-terminal truncated proteins of 37 kDa and 61 kDa respectively. The full length and the truncated proteins contain a proteolytic activity when assayed by SDS/PAGE in two dimensions. The internally deleted protein does not maintain the proteolytic activity. The proteolytic activity was shown not to be the result of non-enzymatic cleavage. A general serine proteinase inhibitor eliminates the proteolytic activity of the full length human and D. melanogaster hsp70. This evidence shows that the proteolytic activity is directly connected to hsp70.

A heat stable protease was identified as the cause of textural degradation in cooked arrowtooth flounder (Atheresthes stomias) muscle. Maximum proteolytic activity in the fish muscle was observed between 55°C and 60°C and myosin heavy chain appeared to be the primary substrate for the enzyme. Degradation of this myofibrillar protein at 55°C was extremely rapid and myosin heavy chain was completely hydrolyzed to peptide fragments smaller than actin, while actin itself was unaffected. A single strand 32kD proteolytic enzyme was extracted from the muscle and purified 125-fold. The enzyme was stable to freezing for up to 6 months. Activity of the semi-purified enzyme at 55°C was optimal against casein between pH 6.0 and 7.0. Sulfhydryl reagents p-chloromercuriphenylsulfonic acid, iodoacetate, iodoacetamide and cystatin were effective in inhibiting enzyme activity in casein assays. The serine protease inhibitors phenylmethylsulfonylfluoride and trypsin-chymotrypsin inhibitor appeared to activate enzyme activity against casein. Adenosine triphosphate was also an activator. Arrowtooth flounder was then considered as a raw material for surimi, since the surimi process provides for repeated washing of the minced muscle and a final mixing step during which inhibitory substances can be conveniently added. Arrowtooth muscle was monitored at all stages of surimi production. There was no evidence of myosin degradation on sodium dodecyl sulphate polyacrylamide electrophoretic gels at any time during surimi production or during the preparation of samples for testing. However, when the washed mince was incubated at 55°C, 12% residual proteolytic activity was observed. This level was sufficient to degrade the myosin component of surimi gels prepared from the control surimi to which no inhibitors had been added. The food grade substances tested for proteolytic inhibition were bovine blood plasma powder, egg white powder, whey protein concentrate, carrageenan and crude α₂-macroglobulin. Addition of plasma and/or egg white powders to control surimi resulted in a product that was comparable to pollock in functional properties as measured by gel strength, expressible moisture and fold tests. Electrophoretic comparison of surimi made with 1.0% or 2.0% plasma powder or egg white with surimi produced with 0.1% or 0.2% α₂-macroglobulin suggested that the plasma and egg white contributed gel enhancing effects in addition to protease inhibition. Carrageenan was not effective as either a protease inhibitor or gel enhancer. / Graduation date: 1992

This thesis examines the design, synthesis and biological applications of a series of inhibitors which incorporate an azobenzene photoswitch, a peptidyl backbone and a trifluoromethyl ketone warhead. The photoswitch can be isomerised by irradiation with UV or visible light and has been employed to modulate the reactivity of the enzyme. Chapter one gives a brief outline of some of the important areas related to this work. Examples of previously utilised photoswitches as well as some background on serine protease and the uses of fluorine in medicine has been covered. Chapter two outlines the synthesis of the key trifluoromethyl carbinol 2.6 by two different methods. The condensation of a fluorinated aldehyde with a nitroalkane affords an α-nitro trifluoromethyl carbinol which can be reduced to give the desired amine 2.6. Treatment of oxazolones with trifluoroacetic anhydride via a modified Dakin-West reaction gives trifluoromethyl ketones which can be reduced to give trifluoromethyl carbinols. Chapter three investigate the synthesis of substituted stilbenes and phenanthrenes as alternative molecular switches to azobenzenes. Molecular modelling of phenanthrenes suggests they may be suitable mimics of E-azobenzenes. Chapter four outlines the synthesis of a series of mono and disubstituted azobenzenes by direct sulfonation of azobenzene or by condensation of nitroso arenes with aryl amines. The switches incorporate one or two peptidyl residues designed to improve specificity towards the enzyme. Chapter five examines the photoisomerisation of eight potential inhibitors by irradiating with UV or visible light. Irradiation with UV light enriches the sample to give 78-93 % of the Z-isomer. Irradiation with visible light gave photostationary states with 14-21 % Z-isomer. Ambient photostationary states are ca. 22 % Z-isomer. Chapter six looks at the testing of five trifluoromethyl ketones as potential inhibitors ofα-chymotrypsin. The inhibitors vary in substituents, substitution patterns and chain length. The inhibitors were tested at both ambient and Z-enriched photostationary states and were found to exhibit slow binding kinetics. In all cases the Z-enriched inhibitor solution was at least 3-fold more potent than the ambient solution. Chapter seven is an experimental chapter and outlines the synthesis of the compounds prepared in this thesis.

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This thesis presents the first example of reversible photoregulation of the binding of a protease, α-chymotrypsin, to a surface. A modular approach is used involving the azobenzene photoswitch group, a surface linker and an enzyme binding group. This approach is designed to be easily extended to the photoregulation of binding of other proteases to surfaces by use of enzyme binding groups selective to these proteases. Chapter one gives a brief outline of some of the important areas involved in to this work, including molecular switches, proteases and surface modification. Chapter two describes the synthesis of azobenzene-containing boronate esters designed as photoswitch inhibitors of α-chymotrypsin. Boronate esters were prepared containing the aminophenylboronate group or the peptidomimetic borophenylalanine group for enzyme binding and a range of substituents designed for enzyme affinity and/or surface attachment. Syntheses primarily involved peptide coupling reactions and azobenzene formation by condensation of nitrosobenzenes and anilines. Coupling reactions were successfully carried out using EDCI or isobutyl chlorofomate in several cases where other reagents gave unacceptable decomposition. Chapter three describes the syntheses and HPLC stability studies of derivatives of a noncovalent α-chymotrypsin inhibitor. Several dipeptide-based compounds containing either an amide group for surface attachment or an azobenzene group for photoswitching were prepared, primarily using peptide coupling reactions. Each compound was incubated with α-chymotrypsin to assess its stability, and all were found by HPLC monitoring to be stable to α-chymotrypsin catalysed hydrolysis. Chapter four describes syntheses of azobenzene-containing trifluoromethylketones and α-ketoesters designed as photoswitch inhibitors of α-chymotrypsin. Trifluoromethylketones/α-ketoesters containing amine groups for surface attachment were prepared, primarily using peptide coupling reactions, but could not be isolated due to the incompatibility of the electrophilic ketone and primary amine groups. Trifluoromethylketones/α-ketoesters containing terminal alkynes for surface attachment were prepared either by the attachment of an alkyne substituent group to a symmetrical azobenzene core or by Pd-catalysed reaction of a protected alkyne with an azobenzene having a halide substitutent. Chapter five describes syntheses of sulfur-containing surface linkers for use in surface attachment of the photoswitch inhibitors described in chapters 2-4. A range of compounds containing disulfide or protected thiol groups for surface attachment and azide or carboxylic acid groups for inhibitor attachment were prepared. Syntheses primarily involved coupling of functionalised alcohols/amides to carboxylic acid-containing disulfides/thioacetates. Selected linkers were attached to azobenzenes by amide coupling or azide-alkyne cycloaddition for surface attachment, photoswitching and/or enzyme assay. Azide-alkyne cycloaddition yields were initially poor, but were improved by use of stoichiometric amounts of copper catalyst. Chapter six describes UV/vis photoisomerisation studies and enzyme assays carried out to assess enzyme photoswitching of the compounds described in chapters 2-5. The trifluoromethylketones and α-ketoesters described in chapter 4 gave the best results, with moderate inhibition of α-chymotrypsin (µM affinity constants) and up to 5.3 fold changes in inhibition on UV/vis irradiation. Many of the boronate esters described in chapter 2 were found to inhibit α-chymotrypsin, but were somewhat unstable to irradiation. The dipeptide-based compounds described in chapter 3 were inactive against α-chymotrypsin. Good photoisomerisation was obtained for an azobenzene containing a symmetrical disulfide surface linker and poor photoisomerisation was obtained for an azobenzene containing a lipoic acid surface linker. Chapter seven describes surface attachment of selected photoswitch inhibitors and studies of photoregulated enzyme binding to the resultant functional surfaces. Self assembled monolayers (SAMs) of disulfides were formed on gold surfaces and characterised by electrochemistry and contact angle measurements. Binding of α-chymotrypsin to SAMs containing a photoswitch inhibitor was detected by quartz crystal microbalance (QCM), but was found to be largely irreversible. An alkyne-containing photoswitch inhibitor was attached to a surface plasmon resonance (SPR) chip in a two step procedure involving generation of an azide modified surface followed by azide-alkyne cycloaddition. Binding of α-chymotrypsin to the resultant modified surface was detected by SPR and successfully regulated by UV/vis irradiation. Chapter eight provides conclusions for the work described in this thesis and suggests future directions. Chapter nine gives experimental details for the work described in this thesis.

The components involved in an immunoassay were investigated in order to improve the detection limits of the ELISA and to make the assay adaptable to a flow injection analysis (FIA) configuration. The goal being the total automation of the ELISA procedure which is long, tedious and has high standard deviation. The antibody purification and cleavage methods were studied with special emphasis on obtaining products with highest immunological activity. The antibody-enzyme coupling reactions using homobifunctional reagents and heterobifunctional reagents were studied in order to attempt the preparation of highly characterized reagents. The fragments of IgG were coupled to polymeric supports via the hinge thiol groups to retain the maximum immunological activity. This method was found to be superior to those methods involving coupling via amino group. These reagents were used in the development of a sandwich ELISA for bovine IgG. The range of assay was in the 20-1000 femtomole range with a linear dynamic range of 2 orders of magnitude and an accuracy of 2-5%. A competitive ELISA based on the use of immobilized anti-human IgG Fab' fragments was developed. The linear dynamic range for this assay was found to be less than one order of magnitude. The detection limit was in the low picomole range with an accuracy of 2-5%. Based on the principle used in the two assays an enzyme immobilization scheme was developed for the reversible immobilization of these enzymes. Which was subsequently utilized in the determination of substrate in the picomole range in a reagent less FIA technique. The goals of this research project were realized in that the FIA system utilized in this work was capable of carrying out totally automated ELISA assays with an accuracy far surpassing the conventional plate ELISA assays.

This study elucidates the temporal expression and regulation of transglutaminase (TGase) and ornithine decarboxylase (ODCase) during cell proliferation and differentiation. In synchronized CHO cells, there were two peaks of TGase activity expressed in G₁ and a smaller peak of activity in mid S phase. ODCase exhibited a single peak of expression in mid G₁ which was inhibited by the administration of both cycloheximide and actinomycin D. In contrast, the increase in TGase activity was not inhibited at any time measured by administration of either cycloheximide or actinomycin D to these cells. TGase activity in CHO cells was not affected by the addition of analogs of cyclic AMP, whereas ODCase activity was increased at all times measured. Retinol administration increased TGase activity 1 hr after release in CHO cells and the activity remained elevated for 4 hr. Retinol administration resulted in the inhibition of ODCase expression in these cells. The administration of α-melanocyte-stimulating hormone (MSH) to mouse melanoma cells resulted in a biphasic increase of TGase activity and a single peak of ODCase activity within 7 hr. In melanoma cells, addition of cycloheximide abolished the first peak of TGase activity but not the second peak. Actinomycin D did not inhibit either peak of TGase expression. The administration of both cycloheximide and actinomycin D inhibited ODCase activity after MSH stimulation. Analogs of cyclic AMP, when added to log phase mouse melanoma cells, increased ODCase but not TGase activity at all points measured. In these cells, retinoic acid plus MSH markedly enhanced the activity of the initial TGase peak compared to MSH alone. ODCase expression was attenuated with retinoic acid plus MSH. Dexamethasone (DEX) induced the first peak of TGase activity but not the second peak seen with MSH administration alone. Administration of DEX resulted in a peak expression of ODCase activity approximately 30% of that seen with MSH alone. In general, chelation of extracellular calcium with EGTA totally blocked ODCase expression with MSH, retinoic acid or DEX. Partial or total ablation of TGase expression was seen with addition of MSH or retinoic acid, but very little inhibition of this enzyme was evident when EGTA was added with DEX or DEX plus MSH. Addition of calcium after all CA⁺⁺-blocks restored the expression of both enzymes.