The saliva of hematophagous ectoparasites contains a cocktail of vasodilators, anticoagulants and immunosuppressors that maintain blood in a liquid state at the site of the lesion and evade the host's defense mechanisms in suppressing the immune response. Since ticks have evolved to utilize mammals as a source of food, our understanding of the tick material, especially the salivary glands will enhance the control of tick infestation and allow the exploitation of the tick's natural resources. SGE protease activity was determined by measuring the degradation of azocasein. Proteolytic activity was found in the pH range of 3 to 11 with the highest activity at pH 9 followed by pH 7. At pH 3-5 the activity was mainly due to aspartic proteases, whereas at pH 7-9 the activity was due to the action of metallo- and serine proteases. At pH 11, the activity was mainly ascribed to metallo- and aspartic proteinase activity The fibrinogenolytic activity was determined by incubating human fibrinogen in the presence of SGE and monitoring the fibrinogen degradation by SDS-PAGE. SGE degraded the Au-chain of fibrinogen within 2 hours of incubation and even after 24 hours incubation there was no hydrolysis of the Bβ and γ-chains of fibrinogen. Characterization of the fibrinogenolytic activity revealed that metalloprotease activity was present over pH range of 3-9 and at pH 3-5, the cysteine proteases were active. No serine protease activity was found under similar experimental conditions. CE-HPLC separation of the SGE revealed three regions of proteolytic activity. Further characterization of the activity containing fractions using protease inhibitors at various pH values showed that the activity associated with region A is mainly due to the presence of aspartic and cysteine proteases in the lower pH range (< 5). Region B was mainly due to the activity of the metallo- and serine proteases, while the activity in region C was mainly due to the metalloproteinases which were more active in the higher pH range (> 9). CE-HPLC separation of SGE resulted in three regions exhibiting fibrinogenolytic activity at pH 7-9. In region A all four enzyme classes were found while in regions B and C, serine, cysteine and metalloproteinases were found to be responsible for the activity. Region A was further purified on the HIC-column and activity eluted in several peaks which after individual application on SE-HPLC column had similar retention times. The pooled samples were analyzed for purity using C5 RP-HPLC and reducing tricine SDS-PAGE and three bands of relative molecular masses 15, 22 and 12 kDa, respectively were found. In an attempt to purify the proteins in region C, four individual CE-HPLC runs were combined and applied to a fibrinogen affinity column. Reducing SDS-PAGE analysis of bound material showed two bands of relative molecular masses of 31 and 39 kDa, respectively. CE-HPLC region C as well as the SGE control was found to disaggregate platelets aggregated by ADP, epinephrine, collagen as well as TRAP. No disaggregation was observed for the saline negative control. The disaggregation is most probably due to the hydrolysis of the fibrinogen cross-linking platelets by the metalloproteinase activity in region C. Understanding of the proteolytic activities present in the salivary gland and therefore identifying molecules crucial for tick feeding could aid in the development of experimental vaccines. Even though the fibrinogenolytic activity was not purified to homogeneity, this study has laid the groundwork for further experiments in this field.
Dissertation (MSc (Biochemistry))--University of Pretoria, 2006.