Purification and characterization of fluorescent probes from blue green algae
Issued Date
2024
Copyright Date
1990
Resource Type
Language
eng
File Type
application/pdf
No. of Pages/File Size
xi, 110 leaves : ill.
Access Rights
open access
Rights
ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (M.Sc. (Biochemistry))--Mahidol University, 1990
Suggested Citation
Luckana Laousoontornsiri Purification and characterization of fluorescent probes from blue green algae. Thesis (M.Sc. (Biochemistry))--Mahidol University, 1990. Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/101033
Title
Purification and characterization of fluorescent probes from blue green algae
Alternative Title(s)
การเตรียมและการศึกษาคุณสมบัติของสารฟลูออเรสเซนต์จากสาหร่ายสีน้ำเงินแกมเขียว
Author(s)
Advisor(s)
Abstract
Phycocyanin which is a protein component of phycobilisomes has been purified from Spirulina platensis. It is an effective fluorescent protein with high quantum yield. Phycocyanin is gaining wide acceptance for use as fluorescent probe in various different analytical techniques. The cyanobacteria Spirulina platensis is a type of blue-green algae found as natural bloom in water environment. It has ability for CO(,2) fixation using the light harvesting phycobili some proteins in photosynthesis process. Phycocyanin was found as major components upon disruption of spirulina platensis by repeated freeze-thawing of the cell mass. The blue-green algae is abundant in natural water source and serves as a suitable staring terial. The deep blue supernate after removal of cell debris contained large amount of phycocyanin. A large scale preparation and purification procedure for phycocyanin was developed in this study. The protein was purified from DEAE cellulose collum chromatography by linear salt gradient elution. It has characteristics of specific absorbance at A620 which was used for monitoring the phycocyanin. The protein was eluted from the column at 0.15 M NaCl as almost pure phycocyanin peak. The absorbance ratio A620/A280 of fractions greater than 3.4 was found to be homogeneous phycocyanin and used as criteria for purity of the protein. Batch binding of phycocyanin with DEAE cellulose followed by step gradient salt elution was empioyed in large scale purification as modified and adapted from the collum method. The fluorescent properties of phycocyanin was characterized with maximum emission at 640 nm. It was sensitive to temperature above 50 C and pH8. The optimum ranges for fluorescence were 20C 40C and pH 4-8. Phycocyanin was also found sensitive to concentration quenching which is a typical nature of fluorophores. Subunit molecular weight was 17.5 kd and the native phycocyanin had molecular weight 208 kd, indicating the phycocyanin was multimeric protein containing 12 subunits in the active from. Avidin was also prepared and purified from egg-white in this investigation for conjugation study with phycocyanin. Large scale purification of avidin from diluted egg-white solution was developed by modification of the previously reported methods. The purification procedure was divided into two steps. The first step was batch binding of avidin with CM-cellulose followed by extensive washing and step wise elution with ammonium carbonate. Detection of avidin was monitored by specific dye binding assay using 4-hydroxyazobenzene 2-carboxylic acid (HABA) which displayed absorbance of A500. the second step employed 2-iminobiotin agarose as affinity column for purification of avidin eluted from CM-cellulose. Combination of two steps resulted in 74% recovery of purified avidin with a specific activity increased from 4.1 (ug biotion/g protein) ad CM-cellulose step to 15.7 in the final affinity step. The molecular weight of purified avidin was 18.5 kd as reported in this study. Conjugation of phycocyanin and avidin was carried out using different chemicals modifying method. Glutaraldehyde and carbodiimide were found unsatisfactory and no phycocyanin-avidin conjugate was resulted in these methods. However, the heterobifunctional reagent, SPDP, was found satisfactory for the formation of phycocyanin-avidin conjugate. The conjugate had both A620 property of phycocyanin and A500 for HABA binding to avidin in the same compels. The conjugate with molecular weight 37 kd probably represented 1:1 ratio and 1:2 ratio for the 55 kd conjugate. This study demonstrated that it was possible to perform large scale preparation of phycocyanin and avidin from simple and abundant starting materials as well as the utilization of the two biomolecules for analytical and practical purposes.
Description
Biochemistry (Mahidol University 1990)
Degree Name
Master of Science
Degree Level
Master's degree
Degree Department
Faculty of Science
Degree Discipline
Biochemistry
Degree Grantor(s)
Mahidol University