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Title: Proteolysis using enzymes in vitro
Author: Bradbear, N. J.
ISNI:       0000 0001 3474 4719
Awarding Body: Durham University
Current Institution: Durham University
Date of Award: 1982
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A variety of methods were tested for their ability to completely hydrolyse proteins to free amino acids in vitro. The most successful method was found to be that using the enzyme mixture papain, prolidase and aminopeptidase-M, which together completely hydrolysed the test substrates ovalbumin, BSA and insulin (B chain). A range of enzymes were coupled to insert supports, and although such immobilization simplified handling of the enzymes, the coupled enzymes proved less efficient for proteolysis than their soluble counterparts. Amino acid analysis and SDS polyacrylamide gel electrophoresis were used to identify the end products of proteolysis. The use of a pH-stat was found unsuitable for the measurement of proteolysis. In vitro proteolysis was used to study the digestibility of glycoprotein II, purified from Phaseolus vulgaris. Denatured glycoprotein II was fully digested by the enzymic method, although in its native form, the glycoprotein was only partially digested. The digestibility of trypsin inhibitor, purified from Phaseolus vulgaris was also studied. Both in its native form, and after heat treatment, the trypsin inhibitor was poorly hydrolysed. It was also found that native trypsin had some resistance to digestion conferred upon it by the presence of the inhibitor. Active trypsin inhibitor was detected in the faeces of rats whose diet included the inhibitor. The significance of these findings in vitro and in vivo were discussed. The use of proteolysis in vitro to measure the availability of amino acids was found comparable with standard assays for the measurement of available lysine and methionine.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry