Dunaliella, a genus of unicellular, biflagellate green algae, is one of the most studied microalgae for mass culture and is of commercial importance as a source of natural -carotene. Dunaliella species have the desirable properties of halotolerance and photoautotrophy that makes their large-scale culture simple and cheap using resources unsuitable for conventional agriculture. The ease and cost-effectiveness of culture makes Dunaliella a desirable target for increased production of natural compounds by metabolic engineering or for exploitation as biological factories for the synthesis of novel high-value compounds. However, the lack of efficient genetic transformation systems has been a major limitation in the manipulation of these microalgae. In chapter four we describe the development of a nuclear transformation system for Dunaliella tertiolecta. The gene encoding the phleomycin-binding protein from Streptoalloteichus hindustanus, was chosen as the selectable marker as this protein retains activity at high salt concentrations. To drive expression of the chosen selectable marker, two highly expressed Dunaliella tertiolecta RbcS genes and their associated 5' and 3' regulatory regions were isolated and characterised (chapter three). Dunaliella transformation cassettes containing the RbcS promoter and terminator regions flanking the ble antibiotic resistance gene were constructed. These expression cassettes were tested in Chlamydomonas reinhardtii cells and found to drive expression of the ble gene in this heterologous system. This study also demonstrated that truncation of both the D. tertiolecta RbcS1 and RbcS2 regulatory regions significantly increases the expression of the ble gene in C. reinhardtii cells. To determine if the foreign DNA could stably integrate into the Dunaliella genome, four transformation methods: microprojectile bombardment, glass bead-mediated transformation, PEG-mediated transformation and electroporation were tested and a number of parameters varied. Southern blot analysis revealed that the plasmid DNA transiently entered the Dunaliella cells following electroporation but was rapidly degraded. Following electroporation, one stably transformed Dunaliella line was recovered. This is the first demonstration of the stable transformation of this alga. Chloroplast transformation is becoming a favoured method for the production of recombinant proteins in plants, as levels of heterologous protein are often higher than those achieved by transforming the nucleus. The Dunaliella chloroplast genome has not been genetically characterised, and thus there were no existing promoter and terminator sequences or sequences of intergenic regions that could be used for vectors in transformation of the chloroplast. Therefore, this study aimed to isolate and characterise promoters of highly expressed genes and matching terminators capable of driving transgene expression, and also to characterise intergenic regions that would be suitable insertion sites for the vector construct (chapter five). The complete gene sequence of two highly expressed Dunaliella chloroplast genes psbB and rbcL including the promoter and terminator regions as well as the coding sequence of the psbA gene were cloned and sequenced. In addition, the psbA gene is useful as a selectable marker as introduced mutations confer resistance to the herbicide 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU). Two homologous transformation constructs based on mutated psbA genes were developed and tested using microprojectile bombardment. A number of parameters were tested including: the size of the gold microprojectile particle, the distance of the plates from the point of discharge, plating onto membranes or filter paper, helium pressure, addition of an osmoticum to the medium and recovery time. Although no chloroplast transformants were recovered in this study, these homologous recombination constructs should prove useful in the development of a chloroplast transformation protocol. The other major component of this study was to investigate the use of microalgae as an expression system for the production of recombinant proteins. Transformation of Chlamydomonas reinhardtii, a species related to Dunaliella, is well developed. In chapter six, this study examined the expression of two human proteins, -lactalbumin and IGF-1 in Chlamydomonas reinhardtii. Plasmids containing the C. reinhardtii RbcS2 promoter upstream of the cDNAs of these two proteins were introduced into C. reinhardtii cells using glass-bead mediated transformation. Transgenic C. reinhardtii lines were generated and shown to contain the transgenes by PCR and Southern hybridisation. RT- PCR and northern hybridisation were subsequently used to demonstrate that the transgenes were transcriptionally active. The transcripts however, could only be detected by RT-PCR indicating that the genes were transcribed at low levels. Accumulation of the -lactalbumin protein could not be demonstrated, suggesting that although the transgenes were transcribed, they were either not translated or translated at levels below the sensitivity of western blot analysis or that any protein produced was rapidly degraded. Previous studies have indicated that in microalgae codon usage is vital in translation of the foreign protein. Codon modification of the IGF-I and -lactalbumin genes should lead to higher levels of protein accumulation. This study reports the first successful stable nuclear transformation of Dunaliella tertiolecta. Therefore it is now feasible that Dunaliella can be examined as a bioreactor for the expression of recombinant proteins. In addition, two chloroplast genes (psbB and rbcL) and their corresponding promoters and terminators have been characterised and a selectable marker cassette based on the mutated psbA gene constructed.

Whey is a by-product of cheese manufacture, containing a mixture of proteins of commercial value, each having unique attributes for nutritional, biological and food ingredient applications. A tremendous amount of whey, normally treated as a waste product, is produced worldwide each year. This work describes the cation-exchange adsorption of the two major whey proteins, alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) with the purpose of optimising a process for isolating them from whey. Adsorption of pure BLG and ALA was studied onto SP Sepharose FF using 0.1M acetate buffer. Batch experiments were carried out at various pH values for ALA and BLG, and the relevant Langmuir isotherm parameters, dissociation constant, Kd, and maximum binding capacity, qm, were determined. The optimum pH for separation was chosen to be pH 3.7. At pH 3.7, both Kd and qm pertaining to ALA were found to have higher numerical values than those of BLG, implying different characteristics of adsorption of the two proteins on this adsorbent. The Kd for the former protein was almost four times larger than the latter, while qm was 1.3 times higher. Packed-bed column adsorption was performed using a 1-ml column at pH 3.7, flow rate 1 ml/min and initial concentration of 3 mg/ml for BLG and 1.5 mg/ml for ALA both in 0.1M sodium acetate buffer. The t1/2 for the resulting ALA breakthrough was 75% longer than its BLG counterpart. The above results suggest the possibility of the occurrence of competitive adsorption between the proteins when adsorbed simultaneously. In traditional batch uptake experiments, the kinetic rate constants of ALA and BLG in both the single- and two-component systems were determined using the simple kinetic model. The values so obtained implied that BLG was adsorbed faster than ALA. In the confocal laser scanning microscopy experiments, the different behaviour of ALA and BLG in the single-component system with regard to their penetration within the adsorbent beads suggested that the two proteins have different transport mechanisms governing their adsorption. The two-component system results showed that ALA was able to displace BLG in spite of the lower affinity of the former protein to the adsorbent. The packed-bed adsorption and elution of a mixture of ALA and BLG were then investigated under the above conditions but using a 5-ml column. BLG breakthrough occurred first, and its concentration in the outlet exceeded its feed value by 1.6 fold before declining to the feed value, followed by the breakthrough of ALA. ALA displaced and eluted all the BLG from the column in a pure form. Pure ALA could then be eluted with good recovery. The single- and two-component breakthrough curves for ALA and BLG were simulated by the simple kinetic model using the isotherm parameters, but the overshoot phenomenon could only be predicted after correcting these parameters. The evidence of the competitive nature of adsorption observed in binary mixtures was used to develop a facile separation procedure for the two proteins from aqueous solutions of whey concentrate powders. A novel consecutive two-stage separation process was developed to separate ALA and BLG from whey concentrate mixtures. Almost all the BLG in the feed was recovered, with 78% being recovered at 95% purity and a further 20% at 86% purity. In addition, 67% of ALA was recovered, 48% at 54% purity and 19% at 60% purity. The correction factors employed for the pure binary mixture were used to simulate the breakthrough curves of the two proteins in experiments conducted with whey concentrate in each of the two stages of the novel separation process, and there was agreement between the experimental and theoretical results.

Le repliement des protéines est un des problèmes centraux de la biologie. Il s'agit de comprendre comment la chaîne polypeptidique d'une protéine se replie pour acquérir sa structure tridimensionnelle biologiquement active. Il a été démontré dans les années 60 que la forme repliée de la protéine est le plus stable d'un point de vue thermodynamique et qu'il est défini par la structure primaire. La réaction de repliement correspond ainsi à la dernière étape de l'utilisation de l'information contenue dans l'ADN. Cependant, Il est possible que les protéines se replient mal et interagissent entre elles pour former des fibres amyloïdes. Ce sont des agrégats structurés impliqués dans plusieurs maladies comme la maladie d'Alzheimer, de Parkinson... <br>Ces phénomènes sont étudiés ici dans le cas de l'alpha-lactalbumine, une protéine du lait qui possède un site de liaison pour le calcium. Le repliement est tout d'abord étudié en présence de métaux se liant au site du calcium. Ces expériences sont couplées à des expériences de dénaturation thermiques pour caractériser le rôle de la fixation des métaux sur les différents états de la protéine et son influence sur la cinétique de repliement.<br>La réaction est ensuite caractérisée en absence d'ion métallique. Elle est alors beaucoup plus lente et complexe. Différentes techniques spectroscopiques sont utilisées. Les résultats obtenus permettent de proposer un schéma réactionnel selon lequel un état précurseur de fibres amyloïdes est transitoirement peuplé. Enfin, pour compléter cette étude, les effets des interactions entre protéines sur la formation de fibres amyloïdes ont été étudiés pour différentes concentrations en sel.

The effects of peptides derived from β-lactoglobulin and α-lactalbumin on metabolic syndrome were studied. α-lactalbumin and β-lactoglobulin were hydrolyzed with trypsin, alcalase, flavourzyme, or a combination of alcalase and flavourzyme and fractionated. Angiotensin coverting enzyme inhibition of the < 1 kDa fraction of alcalase hydrolyzed β-lactoglobulin was 95 %. Antioxidant activity of the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme was 18 %. Stimulated adipocytes incubated with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with either trypsin or alcalase produced 30 pg/mL of interleukin 6. Adiponectin and glucose transporter type 4 secretions increased 1.1 and 0.86 fold respectively during incubation with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme. Results indicate that β-lactoglobulin peptides formed with alcalase and a combination of alcalase and flavourzyme influence markers associated with metabolic syndrome and may be useful as functional foods or nutraceuticals.

The effects of peptides derived from β-lactoglobulin and α-lactalbumin on metabolic syndrome were studied. α-lactalbumin and β-lactoglobulin were hydrolyzed with trypsin, alcalase, flavourzyme, or a combination of alcalase and flavourzyme and fractionated. Angiotensin coverting enzyme inhibition of the < 1 kDa fraction of alcalase hydrolyzed β-lactoglobulin was 95 %. Antioxidant activity of the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme was 18 %. Stimulated adipocytes incubated with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with either trypsin or alcalase produced 30 pg/mL of interleukin 6. Adiponectin and glucose transporter type 4 secretions increased 1.1 and 0.86 fold respectively during incubation with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme. Results indicate that β-lactoglobulin peptides formed with alcalase and a combination of alcalase and flavourzyme influence markers associated with metabolic syndrome and may be useful as functional foods or nutraceuticals.

We have developed an analytical technique for single molecule force spectroscopy (SMFS) data that avoids filtering prior to analysis and performs pattern recognition to identify distinct SMFS events. The technique characterizes the signal similarity between all curves in a data set and generates a hierarchical clustering tree, from which clusters can be identified, aligned, and examined to identify key patterns. This procedure was applied to alpha-lactalbumin (aLA) on polystyrene substrates with flat and nanoscale curvature, and bacteriorhodopsin (bR) adsorbed on mica substrates. Cluster patterns identified for the aLA data sets were associated with different higher-order protein-protein interactions. Changes in the frequency of the patterns showed an increase in the monomeric signal from flat to curved substrates. Analysis of the bR data showed a high level of multiple protein SMFS events and allowed for the identification of a set of characteristic three-peak unfolding events.

The human &alpha;-lactalbumin (&alpha;-LA) molten globule formed at low pH is a model for the study of protein folding intermediates. The molten globule lacks native-like side-chain interactions, resulting in a fluctuating ensemble of tertiary structures, characterisation of which has been precluded by severe line-broadening in NMR spectra and a lack of long-range NOEs. Paramagnetic relaxation enhancements (PREs) have been measured in a variant of &alpha;-LA in which all native cysteines have been mutated to alanine (all-Ala &alpha;-LA). Cysteine residues have been mutated into regions of interest and spin labelled with MTSL. These measurements have confirmed that all-Ala &alpha;-LA forms a compact molten globule. Transient, long-range interactions that are stabilising the compact fold have also been identified using PREs measured in urea-denatured states. This has identified several interactions formed by hydrophobic residues from both the &alpha;- and &beta;-domain, which could be important for initiating and driving folding. The molten globule’s 3D topology has been probed by measuring long-range distances between MTSL pairs using Double Electron-Electron Resonance (DEER). Broad distance distributions have been identified between elements of secondary structure, indicative of a fluctuating but compact fold. By contrast, a narrower distance distribution has been measured within one of the major helices, indicative of native-like secondary structure. The surface accessibility of all-Ala &alpha;-LA and that of two other variants ([28-111] &alpha;-LA and 4SS &alpha;-LA) has been probed using solvent PREs obtained using TEMPOL, a paramagnetic co-solute. This has revealed differences in the solvent-exposure of hydrophobic residues due to the removal of disulphide bonds. This method has also identified buried hydrophobic residues that contribute to forming the molten globule’s stable, native-like core.

Le lait écrémé est utilisé depuis plus d’un demi-siècle comme diluant protecteur des spermatozoïdes de mammifères. Depuis quelques années, il existe une demande grandissante pour des diluants exempts de produits d’origine animale. Toutefois, le mécanisme par lequel le lait protège les spermatozoïdes n’est pas connu, ce qui rend difficile de lui trouver un substitut. Les protéines majeures du plasma séminal de taureau, les protéines « Binder of SPerm » (BSP), sont néfastes lors de la conservation de la semence. Les spermatozoïdes sont en contact avec une grande concentration de protéines BSP qui stimulent une extraction continuelle de cholestérol/phospholipides de leur membrane plasmique. Les lipoprotéines de faible densité (LDL) du jaune d’oeuf, un autre composé utilisé dans les diluants, empêcheraient les protéines BSP de se lier à la membrane des spermatozoïdes de taureaux et de stimuler un efflux des lipides membranaires, ce qui les protégerait durant la conservation. Notre hypothèse était que les protéines du lait protègent les spermatozoïdes durant la conservation en séquestrant les protéines BSP. Premièrement, nous avons démontré par filtration sur gel qu’il y a une interaction entre les protéines BSP bovines et les protéines du lait. Le lait écrémé a été fractionné en trois fractions : F1 (alpha-lactalbumine, bêta-lactoglobuline et caséine kappa), F2 (toutes les protéines du lait) et F3 (sels, sucres et petits peptides). Les protéines BSP1 et BSP5 ont une affinité plus grande pour F1 que BSP3, tandis que toutes les protéines BSP ont une affinité pour F2. Le titrage calorimétrique isotherme a permis de confirmer l’interaction entre les protéines BSP et les protéines du lait. L’association entre la protéine BSP1 bovine et les micelles de caséines est caractérisée par une constante d’affinité (Ka) de 3.5 × 10^5 M-1 et un paramètre stoichiométrique (n) de 4,5 BSP1 pour une caséine. L’association entre la protéine BSP1 bovine et l’alpha-lactalbumine (une protéine du sérum principale), est caractérisée par un Ka de 2.4 × 10^5 M-1 et une valeur “n” de 0,8. Ces résultats indiquent que le lait protège les spermatozoïdes bovins en séquestrant les protéines BSP grâce à une interaction protéine : protéine, tandis que le jaune d’oeuf les protège grâce à une interaction protéine : lipoprotéine. Deuxièmement, nous avons démontré par filtration sur gel que les protéines homologues aux BSP bovines retrouvées dans le plasma séminal de porc, d’étalon et de bélier ont une affinité avec les protéines du lait, ce qui suggère que le mécanisme de protection des spermatozoïdes par le lait pourrait être le même chez ces espèces. Troisièmement, nous avons caractérisé l’interaction entre BSP1 bovine et les LDL du jaune d’oeuf qui a un Ka de 3.4 ± 0.4 × 10^6 M-1 et une valeur de « n » de 104 BSP1 pour une particule de LDL, indiquant qu’il existe des différences entre le mécanisme de protection des spermatozoïdes par le lait et le jaune d’oeuf. Nous croyons que les résultats présentés dans cette thèse aideront à créer de nouveaux diluants ne contenant pas de produits d’origine animale afin de cryoconserver les spermatozoïdes des mammifères. / Skim milk is being used as a protective agent for mammalian semen conservation over half a century. Recently, there has been increased interest in developing extenders free of animal products. However, it is difficult to find suitable component in order to replace milk as an extender, because the mechanisms by which milk protect sperm against cooling and freezing damages during the storage is unknown. The Binder of SPerm (BSP) proteins are the major proteins of bull seminal plasma and they are harmful during sperm storage. In fact, sperm would be in contact with a large quantity of BSP proteins that induce a continuous cholesterol and phospholipids efflux from the sperm membrane during storage. When bull sperm is diluted with an extender containing egg yolk, another compound frequently used in extender, the low-density lipoproteins (LDL) present in the egg yolk prevent the binding of the BSP proteins to the sperm membrane, thus, preventing the lipid efflux from the sperm membrane induced by the BSP proteins. Our hypothesis was that milk proteins would protect sperm during storage by binding BSP proteins. First, we demonstrated by gel filtration that bovine BSP proteins could bind the milk proteins. Skim milk was fractionated into three fractions: F1 (alpha-lactalbumin and beta- lactoglobulin, the major whey proteins and kappa-casein), F2 (mainly caseins and all other milk proteins in small amounts) and F3 (salts, sugars and small peptides). Bovine BSP1 and BSP5 have more affinity for F1 as compared to BSP3 and all the BSP proteins have affinity for F2. We confirmed the interaction between bovine BSP proteins and milk proteins by isothermal titration calorimetry. The binding of BSP1 to casein micelles is characterized by an affinity constant (Ka) of 3.5 × 10^5 M-1 and of a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin (one of the major whey proteins) is characterized by a Ka of 2.4 × 10^5 M-1 and a “n” value of 0.8. These results support our contention that milk can protect sperm by preventing the BSP proteins’ binding to the sperm membrane attributable to a protein : protein interaction, while egg yolk sperm protection is attributable to a protein : lipoprotein interaction. Second, our studies showed that the homologous BSP proteins found in the boar, stallion and ram seminal plasma can bind the milk proteins. These results indicate that the mechanism of sperm protection by milk in these species should be similar to the one in bovine species. Third, we characterized the interaction between bovine BSP1 protein and LDL from hen’s egg yolk. The binding was characterized by a Ka of 3.4 ± 0.4 × 10^6 M-1 and a « n » value of 104 BSP1 per LDL particle. Our results indicated that there is difference between the mechanism of sperm protection by milk and egg yolk. We believe that the results presented in this thesis may help to create new extenders free of animal product for mammal sperm preservation in liquid or frozen state.

Le lait écrémé est utilisé depuis plus d’un demi-siècle comme diluant protecteur des spermatozoïdes de mammifères. Depuis quelques années, il existe une demande grandissante pour des diluants exempts de produits d’origine animale. Toutefois, le mécanisme par lequel le lait protège les spermatozoïdes n’est pas connu, ce qui rend difficile de lui trouver un substitut. Les protéines majeures du plasma séminal de taureau, les protéines « Binder of SPerm » (BSP), sont néfastes lors de la conservation de la semence. Les spermatozoïdes sont en contact avec une grande concentration de protéines BSP qui stimulent une extraction continuelle de cholestérol/phospholipides de leur membrane plasmique. Les lipoprotéines de faible densité (LDL) du jaune d’oeuf, un autre composé utilisé dans les diluants, empêcheraient les protéines BSP de se lier à la membrane des spermatozoïdes de taureaux et de stimuler un efflux des lipides membranaires, ce qui les protégerait durant la conservation. Notre hypothèse était que les protéines du lait protègent les spermatozoïdes durant la conservation en séquestrant les protéines BSP. Premièrement, nous avons démontré par filtration sur gel qu’il y a une interaction entre les protéines BSP bovines et les protéines du lait. Le lait écrémé a été fractionné en trois fractions : F1 (alpha-lactalbumine, bêta-lactoglobuline et caséine kappa), F2 (toutes les protéines du lait) et F3 (sels, sucres et petits peptides). Les protéines BSP1 et BSP5 ont une affinité plus grande pour F1 que BSP3, tandis que toutes les protéines BSP ont une affinité pour F2. Le titrage calorimétrique isotherme a permis de confirmer l’interaction entre les protéines BSP et les protéines du lait. L’association entre la protéine BSP1 bovine et les micelles de caséines est caractérisée par une constante d’affinité (Ka) de 3.5 × 10^5 M-1 et un paramètre stoichiométrique (n) de 4,5 BSP1 pour une caséine. L’association entre la protéine BSP1 bovine et l’alpha-lactalbumine (une protéine du sérum principale), est caractérisée par un Ka de 2.4 × 10^5 M-1 et une valeur “n” de 0,8. Ces résultats indiquent que le lait protège les spermatozoïdes bovins en séquestrant les protéines BSP grâce à une interaction protéine : protéine, tandis que le jaune d’oeuf les protège grâce à une interaction protéine : lipoprotéine. Deuxièmement, nous avons démontré par filtration sur gel que les protéines homologues aux BSP bovines retrouvées dans le plasma séminal de porc, d’étalon et de bélier ont une affinité avec les protéines du lait, ce qui suggère que le mécanisme de protection des spermatozoïdes par le lait pourrait être le même chez ces espèces. Troisièmement, nous avons caractérisé l’interaction entre BSP1 bovine et les LDL du jaune d’oeuf qui a un Ka de 3.4 ± 0.4 × 10^6 M-1 et une valeur de « n » de 104 BSP1 pour une particule de LDL, indiquant qu’il existe des différences entre le mécanisme de protection des spermatozoïdes par le lait et le jaune d’oeuf. Nous croyons que les résultats présentés dans cette thèse aideront à créer de nouveaux diluants ne contenant pas de produits d’origine animale afin de cryoconserver les spermatozoïdes des mammifères. / Skim milk is being used as a protective agent for mammalian semen conservation over half a century. Recently, there has been increased interest in developing extenders free of animal products. However, it is difficult to find suitable component in order to replace milk as an extender, because the mechanisms by which milk protect sperm against cooling and freezing damages during the storage is unknown. The Binder of SPerm (BSP) proteins are the major proteins of bull seminal plasma and they are harmful during sperm storage. In fact, sperm would be in contact with a large quantity of BSP proteins that induce a continuous cholesterol and phospholipids efflux from the sperm membrane during storage. When bull sperm is diluted with an extender containing egg yolk, another compound frequently used in extender, the low-density lipoproteins (LDL) present in the egg yolk prevent the binding of the BSP proteins to the sperm membrane, thus, preventing the lipid efflux from the sperm membrane induced by the BSP proteins. Our hypothesis was that milk proteins would protect sperm during storage by binding BSP proteins. First, we demonstrated by gel filtration that bovine BSP proteins could bind the milk proteins. Skim milk was fractionated into three fractions: F1 (alpha-lactalbumin and beta- lactoglobulin, the major whey proteins and kappa-casein), F2 (mainly caseins and all other milk proteins in small amounts) and F3 (salts, sugars and small peptides). Bovine BSP1 and BSP5 have more affinity for F1 as compared to BSP3 and all the BSP proteins have affinity for F2. We confirmed the interaction between bovine BSP proteins and milk proteins by isothermal titration calorimetry. The binding of BSP1 to casein micelles is characterized by an affinity constant (Ka) of 3.5 × 10^5 M-1 and of a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin (one of the major whey proteins) is characterized by a Ka of 2.4 × 10^5 M-1 and a “n” value of 0.8. These results support our contention that milk can protect sperm by preventing the BSP proteins’ binding to the sperm membrane attributable to a protein : protein interaction, while egg yolk sperm protection is attributable to a protein : lipoprotein interaction. Second, our studies showed that the homologous BSP proteins found in the boar, stallion and ram seminal plasma can bind the milk proteins. These results indicate that the mechanism of sperm protection by milk in these species should be similar to the one in bovine species. Third, we characterized the interaction between bovine BSP1 protein and LDL from hen’s egg yolk. The binding was characterized by a Ka of 3.4 ± 0.4 × 10^6 M-1 and a « n » value of 104 BSP1 per LDL particle. Our results indicated that there is difference between the mechanism of sperm protection by milk and egg yolk. We believe that the results presented in this thesis may help to create new extenders free of animal product for mammal sperm preservation in liquid or frozen state.

La cartographie peptidique est une méthode qui permet entre autre d’identifier les modifications post-traductionnelles des protéines. Elle comprend trois étapes : 1) la protéolyse enzymatique, 2) la séparation par électrophorèse capillaire (CE) ou chromatographie en phase liquide à haute performance (HPLC) des fragments peptidiques et 3) l’identification de ces derniers. Cette dernière étape peut se faire par des méthodes photométriques ou par spectrométrie de masse (MS). Au cours de la dernière décennie, les enzymes protéolytiques immobilisées ont acquis une grande popularité parce qu’elles peuvent être réutilisées et permettent une digestion rapide des protéines due à un rapport élevé d’enzyme/substrat. Pour étudier les nouvelles techniques d’immobilisation qui ont été développées dans le laboratoire du Professeur Waldron, la cartographie peptidique par CE est souvent utilisée pour déterminer le nombre total de peptides détectés et leurs abondances. La CE nous permet d’avoir des séparations très efficaces et lorsque couplée à la fluorescence induite par laser (LIF), elle donne des limites de détection qui sont 1000 fois plus basses que celles obtenues avec l’absorbance UV-Vis. Dans la méthode typique, les peptides venant de l’étape 1) sont marqués avec un fluorophore avant l’analyse par CE-LIF. Bien que la sensibilité de détection LIF puisse approcher 10-12 M pour un fluorophore, la réaction de marquage nécessite un analyte dont la concentration est d’au moins 10-7 M, ce qui représente son principal désavantage. Donc, il n’est pas facile d’étudier les enzymes des peptides dérivés après la protéolyse en utilisant la technique CE-LIF si la concentration du substrat protéique initial est inférieure à 10-7 M. Ceci est attribué à la dilution supplémentaire lors de la protéolyse. Alors, afin d’utiliser le CE-LIF pour évaluer l’efficacité de la digestion par enzyme immobilisée à faible concentration de substrat,nous proposons d’utiliser des substrats protéiques marqués de fluorophores pouvant être purifiés et dilués. Trois méthodes de marquage fluorescent de protéine sont décrites dans ce mémoire pour étudier les enzymes solubles et immobilisées. Les fluorophores étudiés pour le marquage de protéine standard incluent le naphtalène-2,3-dicarboxaldéhyde (NDA), la fluorescéine-5-isothiocyanate (FITC) et l’ester de 6-carboxyfluorescéine N-succinimidyl (FAMSE). Le FAMSE est un excellent réactif puisqu’il se conjugue rapidement avec les amines primaires des peptides. Aussi, le substrat marqué est stable dans le temps. Les protéines étudiées étaient l’-lactalbumine (LACT), l’anhydrase carbonique (CA) et l’insuline chaîne B (INB). Les protéines sont digérées à l’aide de la trypsine (T), la chymotrypsine (CT) ou la pepsine (PEP) dans leurs formes solubles ou insolubles. La forme soluble est plus active que celle immobilisée. Cela nous a permis de vérifier que les protéines marquées sont encore reconnues par chaque enzyme. Nous avons comparé les digestions des protéines par différentes enzymes telles la chymotrypsine libre (i.e., soluble), la chymotrypsine immobilisée (i.e., insoluble) par réticulation avec le glutaraldéhyde (GACT) et la chymotrypsine immobilisée sur billes d’agarose en gel (GELCT). Cette dernière était disponible sur le marché. Selon la chymotrypsine utilisée, nos études ont démontré que les cartes peptidiques avaient des différences significatives selon le nombre de pics et leurs intensités correspondantes. De plus, ces études nous ont permis de constater que les digestions effectuées avec l’enzyme immobilisée avaient une bonne reproductibilité. Plusieurs paramètres quantitatifs ont été étudiés afin d’évaluer l’efficacité des méthodes développées. La limite de détection par CE-LIF obtenue était de 3,010-10 M (S/N = 2,7) pour la CA-FAM digérée par GACT et de 2,010-10 M (S/N = 4,3) pour la CA-FAM digérée par la chymotrypsine libre. Nos études ont aussi démontrées que la courbe d’étalonnage était linéaire dans la région de travail (1,0×10-9-1,0×10-6 M) avec un coefficient de corrélation (R2) de 0,9991. / Peptide mapping is a routine method for identifying post-translational modifications of proteins. It involves three steps: 1) enzymatic proteolysis, 2) separation of the peptide fragments by capillary electrophoresis (CE) or high performance liquid chromatography (HPLC), 3) identification of the peptide fragments by photometric methods or mass spectrometry (MS). During the past decade, immobilized enzymes for proteolysis have been gaining in popularity because they can be reused and they provide fast protein digestion due to the high ratio of enzyme-to-substrate. In order to study new immobilization techniques developed in the Waldron laboratory, peptide mapping by CE is frequently used, where the total number of peptides detected and their abundance are related to enzymatic activity. CE allows very high resolution separations and, when coupled to laser-induced fluorescence (LIF), provides excellent detection limits that are 1000 times lower than with UV-Vis absorbance. In the typical method, the peptides produced in step 1) above are derivatized with a fluorophore before separation by CE-LIF. Although the detection sensitivity of LIF can approach 10 12 M for a highly efficient fluorophore, a major disadvantage is that the derivatization reaction requires analyte concentrations to be approx. 10 7 M or higher. Therefore, it is not feasible to study enzymes using CE-LIF of the peptides derivatized after proteolysis if the initial protein substrate concentration is <10-7 M because additional dilution occurs during proteolysis. Instead, to take advantage of CE-LIF to evaluate the efficiency of immobilized enzyme digestion of low concentrations of substrate, we propose using fluorescently derivatized protein substrates that can be purified then diluted. Three methods for conjugating fluorophore to protein were investigated in this work as a means to study both soluble and immobilized enzymes. The fluorophores studied for derivatization of protein standards included naphthalene-2,3-dicarboxaldehyde (NDA), fluoresceine-5-isothiocyanate (FITC) and 6-carboxyfluorescein N-succinimide ester (FAMSE). The FAMSE was found to be an excellent reagent that conjugates quickly with primary amines and the derivatized substrate was stable over time. The studied substrates were -lactalbumin (LACT), carbonic anhydrase (CA) and insulin chain-B (INB). The CE-LIF peptide maps were generated from digestion of the fluorescently derivatized substrates by trypsin (T), chymotrypsin (CT) or pepsin (PEP), either in soluble or insoluble forms. The soluble form of an enzyme is more active than the immobilized form and this allowed us to verify that the conjugated proteins were still recognized as substrates by each enzyme. The digestion of the derivatized substrates with different types of chymotrypsin (CT) was compared: free (i.e., soluble) chymotrypsin, chymotrypsin cross-linked with glutaraldehyde (GACT) and chymotrypsin immobilized on agarose gel particles (GELCT), which was available commercially. The study showed that, according to the chymotrypsin used, the peptide map would vary in the number of peaks and their intensities. It also showed that the digestion by immobilized enzymes was quite reproducible. Several quantitative parameters were studied to evaluate the efficacy of the methods. The detection limit of the overall method (CE-LIF peptide mapping of FAM-derivatized protein digested by chymotrypsin) was 3.010-10 M (S/N = 2.7) carbonic anhydrase using insoluble GACT and 2.010-10 M (S/N = 4.3) CA using free chymotrypsin. Our studies also showed that the standard curve was linear in the working region (1.0×10-9-1.0×10-6 M) with a correlation coefficient (R2) of 0.9991.