Covalent Linkage of Proteins to Surface-Modified Poly(organophosphazenes): Immobilization of Glucose-6-Phosphate Dehydrogenase and Trypsin

Harry R. Allcock, Sukky Kwon

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Abstract

Glucose-6-phosphate dehydrogenase (G-6-PDH) and trypsin have been linked covalently to a surface-modified poly[bis(aryloxy)phosphazene] supported on porous alumina particles. Poly(diphen-oxyphosphazene) was surface-nitrated and then reduced to the aminophenoxy derivative. The aminophenoxy sites were then activated by reaction with cyanogen bromide, nitrous acid, or glutaric dialdehyde, followed by treatment with the enzymes in aqueous buffer solutions. The activities of the immobilized enzymes were monitored spectrophotometrically with the use of glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (for G-6-PDH) and N-a-benzoyl-L-arginine-p-nitroanilide (for trypsin). These data were supplemented by an assessment of the total yield of immobilized enzyme by means of a Lowry protein measurement. The maximum immobilization yields (8-10% for G-6-PDH and 50-60% for trypsin) were achieved when the glutaric dialdehyde activated system was used. For both proteins, the immobilized enzyme retained its activity and remained linked to the support through numerous cycles. By contrast, the same enzymes adsorbed on uncoated alumina particles were displaced readily by washing. The storage stabilities of both immobilized enzymes were much greater than those of the free enzymes in solution. A continuous-flow assaying system showed that immobilized trypsin generated a constant activity at three different flow rates. For the G-6-PDH system, enzyme activity and scanning electron microscopy were used to examine the relationship between the substrate topography/surface area and the concentration of immobilized protein molecules.

Original languageEnglish (US)
Pages (from-to)1502-1508
Number of pages7
JournalMacromolecules
Volume19
Issue number6
DOIs
StatePublished - Jan 1 1986

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All Science Journal Classification (ASJC) codes

  • Organic Chemistry
  • Polymers and Plastics
  • Inorganic Chemistry
  • Materials Chemistry

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