Alkaline phosphatase activatable polymeric cross-linkers and their use in the stabilization of proteins.
We report the synthesis of polymeric cross-linking agents, poly(glutamic acid) poly(phosphorothioates), and their use in the cross-linking and stabilization of proteins upon treatment with alkaline phosphatase. We have shown that poly(phosphorothioates) are excellent substrates of alkaline phosphatase, yielding thiolated polymers which react covalently with electrophilic groups introduced into the proteins. Three proteins of different structure and function were cross-linked using this method: calf intestinal alkaline phosphatase, glucose oxidase (Aspergillus niger), and (R)-phycoerythrin. The cross-linking of alkaline phosphatase is self-catalyzed since this enzyme catalyzes the hydrolysis of phosphates, unmasking thiolates which react with the maleimide prederivatized alkaline phosphatase. Incubation of buffered solutions of native alkaline phosphatase at 45 degreesC for 7-14 days resulted in a 35% higher loss of enzymatic activity compared to that of cross-linked enzyme. The effect of cross-linking glucose oxidase is even more notable, ranging from 800% stabilization at 37 degrees C and pH 9.0 to 3000% at 37 degrees C and pH 7.4. (R)-Phycoerythrin cross-linked with 1-3 equiv of poly(phosphorothioates) and incubated at 45 degrees C for 45 days was 20% more fluorescent than the native (R)-phycoerythrin subjected to the same conditions. The stabilizing effect of cross-linking was confirmed by comparing the rate of loss of quaternary structure of the cross-linked (R)-phycoerythrin with that of the native protein.
Bieniarz
C
C
Advanced Drug Delivery, Abbott Laboratories, Abbott Park, Illinois 60064-3500, USA. bienica@hpd.abbott.com
Cornwell
M J
MJ
Young
D F
DF
D000469:Alkaline Phosphatase / Q000737:chemistry*; D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology; D002417:Cattle; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D004795:Enzyme Stability / Q000502:physiology; D005453:Fluorescence; D005949:Glucose Oxidase / Q000737:chemistry; D007422:Intestines / Q000201:enzymology; D015394:Molecular Structure; D010799:Phycoerythrin / Q000737:chemistry; D011099:Polyglutamic Acid / Q000138:chemical synthesis; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature
D003432:Cross-Linking Reagents; D013438:Sulfhydryl Compounds; D010799:Phycoerythrin; D011099:Polyglutamic Acid; D005949:Glucose Oxidase; D000469:Alkaline Phosphatase