Enzymatic activity of alkaline phosphatase inside protein and polymer structures fabricated via multiphoton excitation.
We demonstrate micron scale control of bioactivity through the use of multiphoton excited photochemistry, where this technique has been used to cross-link three-dimensional matrixes of alkaline phosphatase, bovine serum albumin, and polyacrylamide and combinations therein. Using a fluorescence-based assay (ELF-97), the enzymatic activity has been studied using a Michaelis-Menten analysis, and we have measured the specificity constants kcat/KM for alkaline phosphatase in both the protein and polymer matrixes to be on the order of 10(5)-10(6) M(-1) s(-1)and are comparable to known literature values in other environments. It is found that the enzyme is simply entrapped in the polymer matrix, whereas it is completely covalently bound in the protein structures. The relative reaction rate of alkaline phosphatase bound to BSA with the ELF substrate was measured as a function of cross-link density and was found to decrease in the more tightly formed matrixes, indicating a decrease in the diffusion in the matrix.
Basu
Swarna
S
University of Connecticut Health Center, Department of Cell Biology and Center for Biomedical Imaging Technology, Farmington, Connecticut 06030, USA.
Campagnola
Paul J
PJ
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000528:radiation effects; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation / Q000528:radiation effects; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000737:chemistry; D010777:Photochemistry; D017785:Photons; D011108:Polymers / Q000032:analysis / Q000378:metabolism* / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D011506:Proteins / Q000032:analysis / Q000378:metabolism* / Q000528:radiation effects; D011799:Quinazolines / Q000737:chemistry; D052999:Quinazolinones; D012395:Rose Bengal / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry
D009943:Organophosphorus Compounds; D011108:Polymers; D011506:Proteins; D011799:Quinazolines; D052999:Quinazolinones; C079177:2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone; D012395:Rose Bengal; D012710:Serum Albumin, Bovine; D000469:Alkaline Phosphatase