PubMed Health⌕ Search

Biomedical subjects

P V Sundaram

Publications and source records attributed to P V Sundaram.

At least 19 recordsLinked to original sources
PubMed1999

An enzyme thermistor-based assay for total and free cholesterol.

A method to evaluate the free (FC) and total cholesterol (TC) in human serum, bile and gallstone extract using an enzyme thermistor (ET)-based flow injection analysis (FIA) is presented. The cholesterol in high-density (HDL-C) and low density lipoprotein (LDL-C) have also been evaluated. A heparin functionalized Sepharose column was employed for the isolation of HDL and LDL fractions from serum. The estimation of cholesterol and its esters was based on their reaction with cholesterol oxidase (CO), cholesterol esterase (CE) and catalase (CAT). Three different enzyme columns, i.e. co-immobilized CO/CAT (column A), only CE (column B) and co-immobilized CO/CE/CAT (column C) were prepared by cross-linking the enzymes on glass beads using glutaraldehyde. Column A was used for estimating FC and column C was used for estimating total cholesterol (cholesterol plus esterified cholesterol). Column B was used as a pre-column which could be switched 'in' or 'out' in conjunction with column A for the estimation of TC or FC, respectively. A calibration between 1.0 and 8.0 mmol/l for FC and 0. 25 and 4.0 mmol/l for TC was obtained. For more than 2000 assays with the ET device a C.V. of less than 4% was obtained. The assay time was approximately 4 min per assay. The cholesterol estimations on the ET correlated well with similar estimations using a commercially available cholesterol diagnostic kit.

Biosensing Techniques↗
PubMed1998

Modulation of stability properties of bovine trypsin after in vitro structural changes with a variety of chemical modifiers.

Controlled chemical modification of enzymes, targeting groups not involved in the active site, can lead to modified catalysts that are intrinsically more efficient and resistant to heat and denaturing agents. Bovine pancreatic trypsin was covalently modified up to 75-85% with monomeric glutaraldehyde (MGA), polymeric glutaraldehyde (PGA), oxidized sucrose and oxidized sucrose polymers (OSP 70 and OSP 400). Virtually no loss in activity occurred upon modification. Temperature optima of trypsin shifts from 45-76 degrees C and T50 from 54-76 degrees C for the best modified sample made with OSP. The efficiency of the modifiers in stabilization was ranked in the order: OSP 400-T > OSP 70-T > PGA-T > MGA-T > Sucrose-T. Half-life of modified enzymes also followed the same trend. Both stabilization factor and t1/2 decreased with increasing temperatures. The free energy of activation for inactivation delta(deltaG*) varies from 12-20 kJ/mol and the activation enthalpy delta(deltaH*) of the modified trypsin by 80-120 kJ/mol indicating stabilization. Inactivation of modified trypsin by urea is less noticeable. The character of the two-step inactivation process of trypsin changes with the degree of stabilization in that the duration of phase I one increased noticeably as stabilization increases. Native trypsin fluoresces less intensely showing a red shift under the influence of denaturation. Such a fluorescence change is not so obvious for the modified enzymes indicating conformational stability acquired by modification.

Animals↗
PubMed1998

Retardation of thermal and urea induced inactivation of alpha-chymotrypsin by modification with carbohydrate polymers.

Modification of enzymes by means of covalent coupling using soluble polymers results in enzymes which retain high biological activity and display resistance to denaturants, high temperature and chaotropic agents. Alpha-chymotrypsin, which has a potential for use in industrial applications, was covalently modified by reductive alkylation using polymeric sucrose (OSP, molecular weight 70 and 400 kDa), dextran (73 and 250 kDa) and carboxymethyl cellulose (CMC, approximately 12 kDa). The derivatives retained around 50-80% activity depending on the polymer used and the extent of modification. At the same time, they displayed better thermotolerance than their native counterpart with 4-14 degrees C higher T50 values. During thermal inactivation, both the native and modified enzymes showed biphasic inactivation kinetics. Half-life of modified enzymes were 2-66-fold greater for the first phase and 5-250-fold greater than the native for the second phase of inactivation. The activation free energy of inactivation of alpha-chymotrypsin coupled to polymeric sucrose (400 kDa) was 112.85 kJ/mol for the first phase and 114.71 kJ/mol for the second phase, whereas in the case of the native enzyme, the value for the first phase was 101.55 kJ/mol and 103.42 kJ/mol for the second phase. The activation free energy of inactivation (deltaG*), as well as the activation enthalpy values (deltaH*) of all the modified enzymes were greater than those of the native enzyme, which is an indication of stabilization of the protein and a retardation of inactivation that is usually accompanied by unfolding under thermal and chemical stress. The stability of modified alpha-chymotrypsin is in the following order: OSP 400-C > OSP 70-C > CMC-C > Dextran 73-C = Dextran 250-C.

Carboxymethylcellulose Sodium↗
PubMed1997

The first structure at 1.8 A resolution of an active autolysate form of porcine alpha-trysoin.

The first crystal structure of an active autolysate form of porcine alpha-trypsin (APT), a two-chain molecule obtained from the limited autolysis of porcine beta-trypsin at position Lys145-Ser146, has been determined. APT crystallizes in space group P2(1)2(1)2(1) with one protein molecule in the asymmetric unit. The structure was solved by molecular replacement followed by refinement using X-PLOR to an R factor of 0.200 and an R(free) of 0.285 for 8.0-1.8 A data with r.m.s deviations from ideal values of 0.01 A and 1.7 degrees for bond lengths and bond angles, respectively. Comparison with inactive autolysate porcine epsilon-trypsin (EPT) and porcine beta-trypsin in complex with bittergourd trypsin inhibitor (MCT) revealed a small but systematic directional chain shift around the active-site residues from APT to EPT to MCT.

Journal Article↗
PubMed1995

Stability of native and covalently modified papain.

Covalent modification of enzymes with large polymers can produce modified enzymes which retain considerable biological activity and at the same time display resistance to denaturation by high temperatures and chaotropic agents. The cysteine protease, papain, with potential applications in industry, was covalently coupled to polymeric sucrose (mol. wt 400 kDa) at different ratios. The derivatives retained > 80% intrinsic catalytic activity with no change in pH optima and kinetic constants, indicating that the gross tertiary structure was not altered by modification. However, they displayed better thermotolerance than native papain, as indicated by their higher T50 values (6-10 degrees C) and their temperature optima being shifted by 10 degrees C. The half-life of modified papain, calculated from the rate of thermoinactivation, was prolonged by 2- to 30-fold over the native depending on the temperature and proportion of polymeric sucrose in the adducts. The increases in activation free energy of inactivation (1-10 kJ/mol) and activation enthalpy (4-78 kJ/mol) indicate stabilization of the protein and lesser inactivation due to spontaneous unfolding. In the presence of urea, modified papain showed activation, which may be due to a loosening of the 'rigid' structure, reminiscent of the property of thermophilic enzymes.

Enzyme Stability↗
PubMed1993

Borate ion-assisted stabilization of beta-galactosidase from Aspergillus oryzae by polyhydroxy compounds in water-miscible organic solvents.

The stability of beta-galactosidase from Aspergillus oryzae in water-miscible organic solvents in different buffers at various pH values ranging from 4.6 to 8.0 was studied. The stability of the enzyme in all six organic solvents studied was dependent on pH and on the type of buffer ions present. At a given pH, destabilization by organic solvents was highest in sodium borate buffer. The destabilization of beta-galactosidase by these solvents could be reversed by addition of sugars or polyhydroxy compounds exclusively in sodium borate, suggesting a role of borate ions in stabilization. A similar effect of addition of mannitol was observed on deactivation of beta-galactosidase by N, N-dimethylformamide (DMF). Exclusively in sodium borate, at pH 8.0, the addition of mannitol (0.02 M) not only prevented the deactivation by DMF (8%, v/v) but increased the enzyme activity to the level at its optimum pH. Since beta-galactosidase from Aspergillus oryzae is a glycoprotein, complexation of the borate ions to the carbohydrate part may result in change in protein conformation, which, without leading to denaturation or inactivation of the enzyme, may facilitate interaction of the organic solvents with the enzyme leading to its denaturation. Such a denaturation is probably prevented by addition of polyhydroxy compounds, which appear to compete favorably with the carbohydrate moiety of the protein in complexing with borate ions. This should result in the enzyme regaining its native conformation.

Aspergillus oryzae↗
PubMed1988

Routine analysis with immobilized enzyme nylon tube reactors.

The basic strategy involved in the design, development, and application of immobilized enzyme nylon tube reactors for routine analysis is described in this chapter, touching on some of the attractive features of these methods. Extensive data (Tables I and II) and the references provide details which may be needed based on specific methods.

Alcohol Dehydrogenase↗
PubMed1981

Affinity chromatography and immunosorption with acetylcholine receptor attached to nylon tubes.

Nylon-linked proteins were used for affinity trapping and chromatography. As representative examples purified acetylcholine receptor, alpha-cobratoxin and bovine serum albumin were coupled to the activated matrix to serve as biospecific ligands. In particular, acetylcholine receptor was coupled without significant loss of biochemical properties. The resulting affinity tubes bind receptor-specific ligands including immunoglobulins and thus can be used for affinity-chromatographic purposes and immunoassays.

Chromatography, Affinity↗
PubMed1981

Automated determination of cholesterol by use of immobilized aldehyde dehydrogenase.

A method for the fully enzymic determination of cholesterol by use of aldehyde dehydrogenase, immobilized onto nylon-tubing, is described. The method gives good results, correlates well with enzymic cholesterol determination to solution, and is high in precision. More than 4000 tests can be carried out with one reactor, reducing the cost for aldehyde dehydrogenase to a negligible amount.

Aldehyde Dehydrogenase↗
PubMed1980

The use of an immobilized glycerol dehydrogenase nylon-tube reactor in the determination of glycerol.

A continuous-flow system is described that utilizes an immobilized glycerol dehydrogenase nylon-tube reactor integrated into the flow system of a Technicon AutoAnalyzer II for the routine estimation of glycerol in serum and various beverages. Results are compared with determinations made by the solution method that uses the three enzymes glycerokinase, pyruvate kinase and lactate dehydrogenase. These routine trials give reliable and reproducible results with high precision and economy. The glycerol dehydrogenase reactor is stable during intermittent or continuous use for a least 3500 tests. Stored at 4 degrees C when not in use, the reactor is quite stable for several months.

Autoanalysis↗
PubMed1980

Fully enzymic method of plasma triglyceride determination using an immobilized glycerol dehydrogenase nylon-tube reactor.

A new, enzymic method of triglyceride determination in serum and plasma by use of an immobilized glycerol dehydrogenase nylon-tube reactor, integrated into the flow system of an AutoAnalyzer II (Technicon) is described. The combination of this reactor, stable for 1500--2000 tests, with the lipolytic enzymes which are added in solution yields a reliable and reproducible assay, which correlates well with the commonly used fully enzymic triglyceride determination. Using this new method, the cost can be reduced to about one-third of that of the other method.

Animals↗
PubMed1980

Single- and coupled-enzyme nylon-tube reactors for plasma glucose determination with glucose oxidase and aldehyde dehydrogenase.

We describe routine methods for determining glucose in plasma with use of aldehyde dehydrogenase or glucose oxidase-aldehyde dehydrogenase immobilized in a nylon tube that is integrated into a continuous-flow system. Although the coupled-enzyme nylon-tube reactors require the presence of a third enzyme, catalase, in solution, the kinetics are not so complicated as to preclude reliable routine determination of glucose at very low cost. Precision is good, and results correlate well with those by the method involving glucose oxidase in solution. More than 3000 tests may be carried out with one reactor. The immobilized enzymes are stable for several months at 4 degrees C when not in use.

Aldehyde Dehydrogenase↗
PubMed1979

The reversible immobilization of proteins on nylon activated through the formation of a substituted imidoester, and its unusual properties.

Alkylation of nylon produces nylon imidate, which is used for the covalent coupling of enzymes and other proteins and ligands. Nylon imidate is unusually stable when stored wet, with a half-life of about 60 days. Reaction with enzymes has an optimum about pH 7-8 and is extremely rapid, with about 60% of the reaction being complete in the first 10 min. The amidine formed in reaction with an amino group can be displaced by another nucleophile. In view of this finding that nylon amidine is capable of exchanging proteins, it is now concluded that nylon-tube reactors containing immobilized enzymes made by this method may not be used as extracorporeal shunts or as on-line monitors on patients, since the enzyme in the reactor may be released into the circulation by nucleophiles in the blood. This can lead to complications. The chemistry of this displacement reaction is discussed.

Amidines↗