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PRINCIPLES OF SIGNAL GENERATION AND OF COUPLING TO OPTICAL FIBERS: DYNAMIC FLUORESCENCE BIOSENSORS
No full textWe have investigated in detail the transport processes and the enzyme catalyzed
reactions that govern the generation of a fluorescence signal in dynamic biosensors,
using a biosensor for penicillin G with a measurings range of 0,1 — 100 mM
penicillin G. The optical read-out of fluorescence changes is analyzed by considering
the optical power budget. We show the limitations to the final output signal
set by the properties of the source, fibers, optics, filters, detectors, noise, and
fluctuations
BIOSENSORS FOR THE DETECTION OF HEAVY METAL IONS
No full textA concept for the development of biosensors for the detection of heavy
metal ions in aquaeous solution is introduced. Phytochelatins, metallothioneins
and glutathione are used as biological components for the
heavy metal sensors. These peptides/proteins selectively bind heavy
metal ions via thiolate complex formation. The biological component is
immobilized at the surface of an appropriate transducer. Changes within
the layer of immobilized peptides/proteins, effected by binding of
metal ions (e.g. release of protons, changes of mass and optical properties),
are transformed into electrical signals by the transducer
(proton-sensitive field effect transistor, resonating piezoelectric
crystal or optical device).
Preliminary experiments demonstrate the suitability of phytochelatins
and glutathione as biological components for the development of biosensors
for heavy metals
From Flow Injection Analysis to Sensor Injection and Towards New Analytical Methodologies for the Next Century
No full textFlow injection analysis (FIA) has developed within last 15 years
into a well established laboratory technique, the scope of which has
been recorded in several monographs [1 - 5] and in over three
thousand papers published to date. While it would be tempting ( and
comfortable) merely to review this past success, it has been suggested
to me by the Organizer of this Workshop to attempt something more
valuable , that is to look fifteen years into the future and to
speculate how this technique will develop in the hands of the next
generation of our younger colleagues. The futility of such
undertaking is surpassed only by irresistibility of such a task. It is
certain, however that any projection made now, will be surpassed
by actual developments. Indeed, flow injection has already
undergone an amazing transformation from a laboratory technique to
methodology of enhancement of instrumental analysis
CHARACTERIZATION AND BIOTECHNOLOGICAL APPLICATION OF EUBACTERIAL GLYCOPROTEINS
No full textThis contribution summarizes the structural and chemical characterization
of crystalline bacterial cell surface layers (S-layers) and
gives an overview on its biotechnological application potential. These
supramolecular structures represent ideal model systems for learning
how nature has accomplished production and maintainance of structures
at the nanometer level. Crystalline S-layers consist of regularly arranged
protein or glycoprotein molecules and exhibit pores of identical
shape and size in the two-dimensional lattice. This makes them particularly
useful for the production of a completely new type of ultrafiltration
membrane. Chemical modification allows for specific adaptations
of the S-layer, either for ultrafiltration purposes or for coating with
monolayers of specific molecules such as enzymes, antigens or haptens.
These studies have clearly shown that the protein or glycoprotein
molecules are of great interest as patterning elements for nanometer
technologies and in the development of biosensors. S-layers can also be
used as supports for Langmuir-Blodgett films or as carriers for immunomodulating
agents in the development of a new generation of vaccines
STUDIES ON THE STRUCTURE OF CARBOHYDRATE CHAINS OF GLYCOPROTEINS
No full textTo obtain detailed information on the primary structures ofthe carbohydrate chains of glycoproteins,it is
still necessary to degradethe glycoprotein to partial structures each representing only one glycosylation
position. The partial structures suitable for analysis, can be oligosaccharides, oligosaccharide-alditols or
glycopeptides. In our approachfor the analysis of N,O-glycoproteins we cleavefirst the N-linked chains
with the aid of PNGaseF. Theextentofthis reaction has to be checked carefully, in order to makesure that
in case the reaction doesnot lead to complete removal of the carbohydrate chains, the chainsthat are still
linked to the protein do not have a specific structure. Forthis purpose several methodscan be applied. The
pool of N-glycans is separated from the O-glycoprotein, whichis then isolated and subjected to alkaline
borohydride treatment. The pools correspondingto the N- and O-linked chains, respectively, are
fractionated to homogeneous compounds asfar as possible. Analysis of the compoundsis carried out by
500-MHz !H-NMR spectroscopy. The occurrence of non-carbohydrate substituentslike alkyl, acyl, sulfate
and phosphate groups may giverise to serious complications. Often the contents of substituents are far
below molar equivalents in a compound,thereby enhancingthe (micro)heterogeneity. It should be noted
that information about the type andposition of the non-carbohydrate substituents can be obtained by 1D- or
2D-1H-NMRspectroscopy. For the analysis of phosphate-containing oligosaccharides we developed a
1H(31P} relayed spin-echo difference spectroscopy technique to characterize the residue to whichthe
phosphateis attached and to detect the C-atom that is substituted.
By meansof this analytical procedure we were able to determine the structure of glycoprotein-derived
carbohydrate chains for quite a numberof glycoproteins
STRUCTURE DETERMINATION OF N- AND O-GLYCANS USING SOFT IONIZATION MASS SPECTROMETRY
No full textGlycosylation is a crucial event in the post-translational modification
of proteins. Numerous investigations have shown clearly that both the
primary structure and the conformation of glycoproteins are intimately
connected with their biological function and metabolic fate. Many laboratories
are currently directing their efforts towards the development
of more specific and sensitive methods for the determination of the
primary and secondary structure of glycopeptides as a first step, towards
the analysis of their biological function. In view of the complexity
of the structures involved, it is evident that only a combination
of different methods will yield the desired results. In this instance
soft ionization mass spectrometry and high resolution NMR have
proved to be expecially useful in determining such different structural
parameters as, for example, the type and number of sugar components,
the sequence of sugar residues, the sites of the glycosidic linkages
and their anomeric confuguration, the sites of glycosylation on the
peptide chain, and the 3D-conformation of these structures. Glycans
linked N-glycosidically through a chitobiosyl-Asn linkage are usually
released enzymatically from the peptide chain prior to analysis ‚whereas
O-glycans, e.g., of the mucin type with a GalNAc (al-3)-Ser/Thr linkage,
can be released by alkaline borohydride treatment. In this contribution
special emphasis will be given to the potential of mass spectrometric
methods in analysing the complex mixtures that are obtained
after enzymatic release of N-glycans from a single glycosylation site,
or after alkaline borohydride treatment of mucin O-glycans
Remodeling of the Carbohydrate Chains of hCG by Use of Sialyltransferases: Effects on the Biological Activity
No full textHuman chorionic gonadotropin (hCG) is a glycoprotein hormone which contains both N- and O-linked
carbohydrate chains. It consists of two subunits, a and 8. The a-subunit contains two N-linked
carbohydrate chains: a mono-antenna and a non-fucosylated bi-antenna. The $-subunit contains both
(two) N and (four) O-linked carbohydrate structures. Both of the N-linked carbohydrate chainsare biantennary,
one of which is fucosylated
CRYSTALLIZATION AND CHARACTERIZATION OF CANDIDA RUGOSA LIPASE
No full textA lipase isolated from the fungus Candida rugosahas beenpurified and crystallized
in a form suitable for X-ray crystallographic structure determination. Several
proteins with lipolytic activity having isoelectric points from 4.2 to 5.8 were
separated by ion exchange chromatography.The protein having the lowestisoelectric
point was crystallized from 2-methy]-2,4-pentandiol in MES bufferin the presenceof
calcium (II) salts. The crystals with cell dimensions a=64.9(1) 7 b=97.2(1) A
and b=175.8(2)A, grow as large colorless plates often exceeding 0.7 mm in each of
two dimensions. From the diffraction pattern the apparent crystal symmetry is
C222,. Experimental density determination suggests one 60,000 M,. molecule in the
asymmetric unit and approximately 50% solvent by volume. Theratio unit cell volume
to the molecular weightof the contents of the unit cell, Vj, is 2.3 A3/d. Diffraction
is strong to a resolution of 2 A resolution and work is underway to determine
the three dimensional structure of this enzyme
HUMAN GASTRIC LIPASE
No full textNinety percentofthe dietary lipids in humans are triglycerides which constitute the essential part of the 100g to 150g daily
fat intake in industrialized countries. It was thoughtuntil recently that the hydrolysis of dietary triglycerides began in the
intestinal lumen and was catalysed exclusively by pancreatic lipase. Studies on gastrointestinal lipolysis have underestimated
several importantpoints, particularly the role of gastric lipolysis. It is now well established that Human Gastric Lipase (HGL),
is the first lipolytic enzyme involved in dietary fat digestion. HGL originates entirely from the fundic mucosa. Nolipolytic
activity was detected in the lingual, pharyngeal or oesophagus areas. Using immunocytolocalization techniques, cells producing
HGLwereidentified as the chief cells of gastric fundic glands already known to biosynthesize pepsin. HGL was purified to
electrophoretic homogeneity (MW = 50 kDa)from gastric juice. It is a glycoprotein with a glycan moiety amounting about 15
to 20 % ofthe total protein weight. The complete amino acid sequence of HGL,derived from cDNA sequence, shows 80 %
homology with rat lingual lipase. No structural homology exists between human gastric lipase and pancreatic lipase, except the
G-X-S-X-G sequence found in otherlipases andserine esterases. This sequence containsa serine analogousto the essential Ser-
152 in human pancreatic lipase. HGL contains one free sulfhydryl group whichis essential to the expressionoflipaseactivity.
HGL hydrolyses short chain (tributyrin) and long chain (Intralipide) triacylglycerols at similar rates. HGL activity is very
dependentupon the interfacial tension between triacylglycerol and water. In the presence of amphiphiles such as bile salts or
alimentary proteins, the tributyrin-waterinterfacial tension decreases and HGLis activated. Thus HGLis capable of hydrolyzing
triglyceride emulsionsin the presence ofbile salts concentration prevailing in the upper small intestine and in the presence of
alimentary proteins. These observations could explain the high dietary lipid absorption observed underpancreatic lipase
deficiency. In vitro studies showed that prehydrolysis by HGLofIntralipide emulsion enable it to be subsequently hydrolyzed
by humanpancreatic lipase. Fatty acid liberated by HGL probablytriggerthe later action of pancreatic lipase by changing the
interfacial tension
LIPASE FROM PSEUDOMONAS SP.: REACTIONS, CLONING, AND AMINO ACID SEQUENCE ANALYSIS
No full textLipases catalyze reactions not only in water but also in organic
solvents. We have developed several asymmetric organic syntheses using
lipase from Pseudomonas fluorescens in organic solvents. However, all
attempts to acylate [1,1’-binaphthyl]-2,2’-diol with the lipase using
enol esters failed. Among the lipases we screened for this substrate,
lipase from Pseudomonas sp. is the only enzyme that catalyzes both
stereoselective acylation of the diol and deacylation of its esters in
organic solvents. The lipase shows higher activity in the hydrolysis of
triglyceride into glycerol when compared with other lipases. Because of
these characteristic reactivities of the lipase from Pseudomonas sp., we
were interested in the cloning and sequencing of the gene of the enzyme
and amino acid sequence analysis. The gene cloned from Pseudomonas sp.
genome DNAwas 933 base pairs and inserted in plasmid pKK233-2. E. ‘coli
JM109 transformed with the recombinant plasmid showed lipolysis asa
zone of clearing around a colony on agar plate containing tributyrin.
Amino acid sequence deduced from the DNA sequence is similar to that of
lipase from Pseudomonas fragi (47 % identical). Content of serine
residue is unusually high as 12 %. Sequence alignment with other 19
amino acid sequences of lipases suggested that Ser82, His251, and Asp209
are the catalytic residues