SummaryThermodynamic properties of aqueous solutions of both native and modified legumin of broad beans (Viciufuba L.) have been examined. A restricted trypsin-induced proteolysis was used to modify protein structure. Evaluation of protein affinity to ficoll in aqueous solutions showed that modified protein possessed higher hydrophilicity. Thermodynamic properties of diluted solutions were used to predict the phase behaviour in concentrated systems containing protein and ficoll. At specific concentrations of native legumin, the system can separate in two phases, whereas in the case of modified protein the single-phase behaviour of the system was predicted for any concentrations of both components. The experimental data obtained in concentrated systems confirmed predictions of thermodynamic analysis of diluted solutions.
SummaryKinetics and mechanism of proteolysis of broad bean (Viciu ,fubu L.) legumin by trypsin in concentrated solutions at different enzyme-substrate ratios ( E / S = I /40, 1 /loo, 1/200, lj1000) were studied. By the method of HPLC it was established that during proteolysis process ( E / S = 1/40) take place both protein content dccrease in hydrolysate and change of its molecular mass (from 360 to 280 kD), which evidences for the mixed type of proteolysis, including cooperative and non-cooperative mechanisms. Decrease of E / S from 1/40 up to IjlOO-l/lOOO leads not only to unproportional drop in proteolysis rate, but also to decrease of the relative contribution of cooperative proteolysis which is, possibly, connected with presence in protein molecule of at least two groups of attack sites, differentiating by their affinity with enzyme. Change of enzyme-substrate ratio is accompanied also by change of subunit composition of product of non-cooperative proteolysis ('modified protein') and also or temperature and enthalpy of its denaturation.
ZusammenfassungBegrenzte enzymatische Hydrolyse von Legumin aus Ackerbohnen (Viciu fubu L.) durch Trypsin in konzentrierten Losungen. Kontrolle des Hydrolyseprozesses in Abhangigkeit von der Veranderung des Enzym-Substrat-Vcrhaltnisses Es wurden die Kinetik und der Mechanismus der Proteolyse des Legumins von Ackerbohnen (Viciu juhu L.) durch Trypsin in konzentrierten Losungen bei unterschiedlichen Enzym-Substrat-VerhHltnissen ( E / S = 1/40, 1/100, 1/200, I/lOOO) untersucht. Mittels HPLC wurde festgestellt, dal3 wahrend des Proteolyseprozesses ( E / S = 1/40) der Proteingehalt im Hydrolysat abnahm und eine Veranderung der Molmasse (von 360 auf 280 kD) vonstatten ging; dies ist ein Beweis fur eine gemischte Hydrolyse, die kooperative und nicht-kooperative Mechanismen einschlieBt. Eine Abnahme von E / S von 1/40 auf 1/100 bis 1/1000 fiihrt nicht nur zu einer unproportionalen Abnahme der Proteolyserate, sondern auch zu einer Abnahme des relativen Beitrages der kooperativen Proteolyse, die moglicherweise in Zusammenhang steht mit wenigstens zwei Angriffsstellen im Proteinmolekiil, die in ihrer Affinitat zum Enzym differieren. Eine Veranderung des Enzym-Substrat-Verhaltnisses wird begleitet von einer Veranderung der Zusammensetzung der Subeinheiten des Produktes der nicht-kooperativen Proteolyse (,,modifiziertes Protein") und auch der Temperatur und Enthalpie dessen Denaturierung.
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