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USE OF FLOW INJECTION ANALYSIS FOR THE DETERMINATION OF ENZYME ACTIVITIES
No full textAn overview of the most outstanding flow-in jection configura-—
tions used so far for the determination of enzyme activites is
presented. The configurations have been clasiffied into four
groups, namely: simple, stopped-flow, open-closed and HPLC/flowinjection
manifolds
FLOW INJECTION MONITORING OF ENZYME REACTIONS ON SOLID SURFACES
No full textOptosensing flow injection analysis may be used to monitor enzyme reactionson various types of
solid surfaces, e.g., cellulose, Teflon or polypropylene. The surfaces may bein the form ofa pad, a
membraneorparticles (in a column).
While the enzyme maybe in solution, advantages accrueif it is attached to the solid surface.
Traditional enzyme immobilization on surfaces involves chemical bonding reactions, but enzymes may be
directly adsorbed onto certain hydrophobic surfaces.
The principles of optosensing measurements are presented andapplicationsto cellulose pads and gas
barrier membranes for enzyme measurements are reviewed. Recent novel methodsfor derivatizing enzymes
for direct adsorption onto fluorocarbon membranesare presented. A new gas sensing (gas gap) membrane
is described in which the membraneacts as the actual sensor and notjust a barrier, suitable for optosensing
of monitoring enzyme reactions. Preliminary studies of the direct adsorption of urease enzyme onto the
membrane are presented.
Silica based C-18 particles in a microcolumn are used to adsorb an indicator and native urease
enzymé. Optosensing measurementsof urea by measuring transmittance through the particles are presented
AMPEROMETRIC BIOSENSORS BASED ON IMMOBILIZED ENZYMES AND CHEMICALLY MODIFIED ELECTRODES
No full textAmperometric biosensors based on two different reaction mechanisms
are presented. Common to both types is the combination of a selective
catalytic reaction that can be followed amperometrically at 0 mV vs
SCE and below.
One type is based on the chemical modification of carbon pastes with
a dehydrogenase, the necessary cofactor NAD’, and a redox mediator.
In the presence of the enzyme substrate NADH will be produced. The
high overvoltage for the electrochemical oxidation of the NADH is
decreased by the addition of the redox mediator to the paste. The
redox mediators used are phenoxazine derivatives making the
electrocatalytic oxidation of NADH possible at 0 mV vs SCE and below.
A glucose sensor based on glucose dehydrogenase is described.
Another type is based on the co-immobilization of a hydrogen
peroxide producing oxidase with horse radish peroxidase on the
surface of heat-treated graphite. The detection is based on an
apparent direct electron transfer from the electrode to the
immobilized peroxidase starting at +600 mV and reaching a maximum at
about O mV vs SCE. The co-immobilized enzyme layer is stabilised by
the addition of bovine serum albumin and glutaraldehyde to the
reaction mixture. A glucose sensor based on glucose oxidase is
presented
OLIGOSACCHARIDE REPROCESSING OF PLASMA MEMBRANE GLYCOPROTEINS
No full textPlasma membrane glycoproteins of rat liver and cultured rat hepatocytes
may undergo continuous reprocessing of their oligosaccharide
units by de- and reglycosylation of terminal Sugars. In distinct
glycoproteins deglycosylation may also include core Sugars of the
oligosaccharides. As shown for L-fucose, terminal sugars are
presumably removed from the glycoproteins in a prelysosomal compartment.
Both the extent and the rate of terminal deglycosylation are
influenced by cell proliferation and transformation. Subsequent upon
terminal deglycosylation plasma membrane glycoproteins may be refucosylated
and resialylated as has been shown for dipeptidyl peptidase Iv
(DPP IV). Oligosaccharide reprocessing most likely occurs during
recycling of the glycoproteins in between the cell surface and intracellular
compartments
INFLUENCE OF GLYCOSYLATION ON THE FUNCTIONAL PROPERTIES OF HUMAN THERAPEUTIC PLASMA PROTEINS
No full textComparative analysis of the carbohydrate structure of plasma antithrombin III
and recombinant antithrombin III synthesized in Chinese Hamster Ovary cells revealed
differences in the linkage of NeuAc, the presence of higher than biantennary structures
and the presence of proximal fucose. Treatment of the carbohydrate part of
antithrombin III from both sources with glycopeptidase F or sialidase had a strong
negative effect on the serum half-life. In order to analyze the effects of elimination
of individual carbohydrate side chains on the pharmacokinetic and functional
properties of AT III the four N-linked glycosylation sites of the recombinant molecule
were altered individually or in combination by site directed mutagenesis of Asn
to Gln. All mutants showed a shorter serum half-life compared to natural antithrombin
II]. However molecules modified at residues Asn 135, Asn 155 and Asn 192 showed
higher heparin affinity and/or maximal stimulation at lower heparin concentrations.
As in the case of antithrombin III the three N-glycosylation sites of tissue
plasminogen activator mutated individually or in combination. Whereas the specific
activities of single glycosylation mutants were unaltered, simultaneous mutation of
two (Asn 117 and Asn 184) or three Asn residues to Gln resulted in molecules with
2-3 fold higher specific activities
PANCREATIC LIPASE/COLIPASE BINDING SITE INVESTIGATION
No full textPancreatic lipase is responsible for fat digestion in the
intestine. The enzyme, which belongs to a special class of
esterases, realizes an heterogeneous catalysis. In vivo,
because of the strong inhibitory effect of bile salt,the lipase
action requires the presence of a small pancreatic protein,
colipase, the function of which is to anchor lipase to the bile
salt coated lipid interface.
The function of the lipase/colipase system requires the
presence of two topographically distinct binding sites on both
proteins, an interfacial binding site and a protein binding
site. Up to now, only the colipase interfacial binding site is
well documented.
In order to locate the lipase/colipase binding site on each
partner, a covalent cross-linked complex has been obtained
using carbodiimides. Immunological analysis of the complex
clearly confirms the presence of lipase and colipase. The
complex has a Mr (60 kDa) consistent with a stoichiometry of
one mol colipase per mol lipase and retains its catalytic
efficiency towards emulsified substrates.
Moreover, the use of carbodiimides to cross-link lipase and
colipase unambiguously shows the participation of ion-pairing
in the interaction between the two proteins
INHIBITION OF THE LIPASE FROM PSEUDOMONASSPEC. ATCC 21808 BY DIETHYL p-NITROPHENYL PHOSPHATE. HINTS FOR ONE BURIED ACTIVE SITE FOR LIPOLYTIC AND ESTEROLYTIC ACTIVITY
No full textLipases have been shown to beserine-hydrolasessince they are inhibited by serine active reagents
such as phenylmethylsulfonylfluoride (PMSF), boronic acids and organophosphates [1,2,3].
Furthermore, they are surface active enzymes,i.e. they are activated by binding to interfaces [4]
comprising their natural substrates, the micelles of long chain fatty acid triglycerides. It has long
been postulated that lipases undergo a conformational changein this activation process. Recently
it became obvious from x-raystudies [1,5] that the active site is inaccessible to voluminoussubstrates
unless a conformational change occurs sincethe active site is buried undera lid formed by a long
peptide loop.
The lipase from human pancreas presumably hydrolysessoluble substrateslike p-nitrophenyl acetate
(pNPA)at a different site [5]. For the porcine pancreatic lipase it was reported that the C-terminal
peptide fragment (336-449) shows the sameactivity towards pNPA as the complete enzyme but no
activity towards triacylglycerols [6]. Whereas,in the triglyceride hydrolysis Ser-152 is involved [R.
Verger, pers. communication].
Ourinhibition studies suggest that only one active site for both types of substrates exists for the
lipase from Pseudomonas spec. ATCC 21808 and thatthis site is buried
Development of Microbial Sensors for Determination of Xenobiotics
No full textAmperometric biosensors using immobilised microbial cells as the biological component were
developed for the determination of biphenyl and its chlorinated derivatives. Measurements
were based on the respiratory activity of the microbial cells. The influence of different organic
solvents on the respiratory activity was analysed. Different membranes were used for immobilising
the cells across the face of an oxygen electrode; to determine whetherthe polarity of the
membrane had an influence on the substrate degraded
BIOSENSORS FOR THE DETECTION OF HEAVY METAL IONS AND FECAL CONTAMINATION
No full textSensors for the detection of heavy metal ions in waste water and
fecal coliforms in surface water are being developped. Heavy metal
complexing peptides/proteins and antibodies against surface antigens
of fecal coliforms are used as the respective biological components.
As transducers, proton-sensitive field-effect transistors (ISFETs),
mass-sensitive piezoelectric crystals and integrated grating couplers
are investigated
PROPERTIES OF PROTEIN LAYERS AT ELECTRODES
No full textSystematic investigations were carried out concerning reproducible modification procedures
of electrodes with immunoglobulin layers and their influence on electrochemical
reactions. It was found that the inhibition effect depends significantly on the nature of the
electrode process. The results are discussed in view of the intention to measure faradaic
detection reactions at protein-covered electrodes