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controlled immobilization of biotinylated horseradish peroxidase in the development of novel biosensors and bioreactors (avidin). [microform]
controlled immobilization of biotinylated horseradish peroxidase in the development of novel biosensors and bioreactors (avidin). [microform]
상세정보
- 자료유형
- 마이크로피시
- 청구기호
- 저자명
- 서명/저자
- controlled immobilization of biotinylated horseradish peroxidase in the development of novel biosensors and bioreactors (avidin). - [microform]
- 발행사항
- 형태사항
- 153 p. : microfiches ; 11×15 cm.
- 총서명
- UMI Dissertation
- 주기사항
- Source: Dissertation Abstracts International, Volume: 57-11, Section: B, page: 7096.
- 학위논문주기
- thesis (ph.d.)-- - university of kentucky, 1996.
- 초록/해제
- 요약Conventional methods of immobilization result in the random orientation of proteins on the surface of the support. In addition, it leads to the formation of multiple bonds between the protein being immobilized and the support. The random orientations cause unpredictability in the properties of immobilized system, while, multiple-bond formation results in reduced flexibility and activity. These problems can be eliminated by orienting the proteins in a desired manner to control the properties of immobilized protein.
- 초록/해제
- 요약In this work, use of biotin-avidin mediated immobilization for orientation of proteins has been studied. Avidin is an egg white protein with very high affinity for biotin and biotinylated proteins. Biotin moiety can be introduced to a protein by chemical methods, however, preparation of a homogeneous population of biotinylated conjugates is difficult. Horseradish peroxidase (HRP) was chosen as the model enzyme for these immobilization studies, as it has only two reactive lysine residues and preparation of homogeneous population of biotinylated conjugates is possible.
- 초록/해제
- 요약HRP was biotinylated with one hundred molar excess of N-hydroxysuccinimide ester of biotin to obtain a homogeneous population of biotinylated HRP, with two biotin moieties attached. A new method based on a coupled HPLC fluorescence binding assay was developed to determine the extent of biotinylation accurately. The new method is based on the complete hydrolysis of protein by acid hydrolysis or enzymatic digestion to release biotin moiety from the biotinylated protein, followed by the determination of released biotin. This method has excellent sensitivity and selectivity for biotin and does not suffer from steric hindrance problems, common to other methods, and therefore does not underestimate the extent of biotinylation.
- 초록/해제
- 요약A homogeneous population of disubstituted biotinylated HRP was immobilized on carboxylate-modified polystyrene beads coated with avidin (oriented immobilization). The reaction kinetics of this system was compared with the immobilization of HRP by carbodiimide chemistry. A second layer of biotinylated HRP was attached to the oriented immobilized system via avidin-biotin bridges. Crosslinked horseradish peroxidase was also prepared, and the kinetic parameters for the Ping-Pong reaction kinetics of this system studied. The oriented immobilization resulted in an increase in the maximum activity of the HRP and reduced inhibition from hydrogen peroxide compared to the native enzyme. The kinetic parameters of this system were estimated by a modified two-substrate Ping-Pong model.
- 초록/해제
- 요약Biotinylated Cholesterol oxidase (COx) was attached to the oriented immobilized biotinylated HRP to obtain a bienzyme system. The activity of this immobilized system was 32% of the activity free cholesterol oxidase. Immobilization of COx and HRP by carbodiimide chemistry resulted in complete loss of activity. When the order of attachment of biotinylated COx and biotinylated HRP was reversed (avidin-biotin mediated random immobilization) the activity dropped to 16% of the free cholesterol oxidase.
- 복제주기
- Microfiche : UMI . microfiches;11×15 cm.
- 일반주제명
- 일반주제명
- 일반주제명
- 키워드
- 기타저자
- 기본자료저록
- Dissertation Abstracts International. 57-11B.
MARC
008970923s1996 us eng■001MOKWON00234457
■001AAV9713772
■00519991002103921
■008970923s1996 us eng
■035 ▼a(UnM)AAV9713772
■040 ▼aUnM▼cUnM▼dMOKWON
■090 ▼a540▼bR215c
■1001 ▼arao, srivatsa v.
■24510▼acontrolled immobilization of biotinylated horseradish peroxidase in the development of novel biosensors and bioreactors (avidin).▼h[microform]
■260 ▼aU.S.▼buniversity of kentucky▼c1996.
■300 ▼a153 p.▼bmicrofiches▼c11×15 cm.
■350 ▼a$50.6
■44000▼aUMI Dissertation
■500 ▼aSource: Dissertation Abstracts International, Volume: 57-11, Section: B, page: 7096.
■502 ▼athesis (ph.d.)--▼buniversity of kentucky▼d1996.
■520 ▼aConventional methods of immobilization result in the random orientation of proteins on the surface of the support. In addition, it leads to the formation of multiple bonds between the protein being immobilized and the support. The random orientations cause unpredictability in the properties of immobilized system, while, multiple-bond formation results in reduced flexibility and activity. These problems can be eliminated by orienting the proteins in a desired manner to control the properties of immobilized protein.
■520 ▼aIn this work, use of biotin-avidin mediated immobilization for orientation of proteins has been studied. Avidin is an egg white protein with very high affinity for biotin and biotinylated proteins. Biotin moiety can be introduced to a protein by chemical methods, however, preparation of a homogeneous population of biotinylated conjugates is difficult. Horseradish peroxidase (HRP) was chosen as the model enzyme for these immobilization studies, as it has only two reactive lysine residues and preparation of homogeneous population of biotinylated conjugates is possible.
■520 ▼aHRP was biotinylated with one hundred molar excess of N-hydroxysuccinimide ester of biotin to obtain a homogeneous population of biotinylated HRP, with two biotin moieties attached. A new method based on a coupled HPLC fluorescence binding assay was developed to determine the extent of biotinylation accurately. The new method is based on the complete hydrolysis of protein by acid hydrolysis or enzymatic digestion to release biotin moiety from the biotinylated protein, followed by the determination of released biotin. This method has excellent sensitivity and selectivity for biotin and does not suffer from steric hindrance problems, common to other methods, and therefore does not underestimate the extent of biotinylation.
■520 ▼aA homogeneous population of disubstituted biotinylated HRP was immobilized on carboxylate-modified polystyrene beads coated with avidin (oriented immobilization). The reaction kinetics of this system was compared with the immobilization of HRP by carbodiimide chemistry. A second layer of biotinylated HRP was attached to the oriented immobilized system via avidin-biotin bridges. Crosslinked horseradish peroxidase was also prepared, and the kinetic parameters for the Ping-Pong reaction kinetics of this system studied. The oriented immobilization resulted in an increase in the maximum activity of the HRP and reduced inhibition from hydrogen peroxide compared to the native enzyme. The kinetic parameters of this system were estimated by a modified two-substrate Ping-Pong model.
■520 ▼aBiotinylated Cholesterol oxidase (COx) was attached to the oriented immobilized biotinylated HRP to obtain a bienzyme system. The activity of this immobilized system was 32% of the activity free cholesterol oxidase. Immobilization of COx and HRP by carbodiimide chemistry resulted in complete loss of activity. When the order of attachment of biotinylated COx and biotinylated HRP was reversed (avidin-biotin mediated random immobilization) the activity dropped to 16% of the free cholesterol oxidase.
■533 ▼aMicrofiche▼cUMI▼emicrofiches;11×15 cm.
■590 ▼aSchool code: 0102.
■650 4▼aEngineering, Chemical
■650 4▼aChemistry, Analytical
■650 4▼aChemistry, Organic
■653 ▼acontrolled▼aimmobilization▼aof▼abiotinylated▼ahorseradish▼aperoxidase▼ain▼athe▼adevelopment▼aof▼anovel▼abiosensors▼aand▼abioreactors▼a(avidin).
■690 ▼a0542
■690 ▼a0486
■690 ▼a0490
■71020▼auniversity of kentucky.
■7730 ▼tDissertation Abstracts International▼g57-11B.
■790 ▼a0102
■791 ▼aPH.D.
■792 ▼a1996
