Biomedical subjects

D K Chattoraj

Publications and source records attributed to D K Chattoraj.

At least 19 recordsLinked to original sources

PubMed1992

Absolute reaction rate and kinetics of protein adsorption at solid-liquid interfaces.

Extents of adsorption of bovine serum albumin from aqueous solution to the surface of alumina, silica, carbon and chromium powder have been studied as function of time for various values of bulk protein concentration, pH, ionic strength and temperature. The rates of adsorption in all cases have been observed to fit in the first order rate equation with two different rate constants Ka1 and Ka2. Effects of addition of SDS, CTAB and neutral salts on values of Ka1 and Ka2 have also been studied. Using Arrhenius equation the activation energy values Ea1 and Ea2 have been evaluated from the values of Ka1 and Ka2 at three different temperatures, respectively. The corresponding values of enthalpy of activation (delta H*), entropy of activation (delta S*), and free energy of activation (delta G*) have been evaluated using Eyring's equation of absolute reaction rate. The mechanism of protein adsorption has been discussed in the light of basic principles of absolute reaction rate. It has been found that for Ka1 the delta H*1 greater than T delta S*1 and for Ka2 T delta S*2 greater than H*2, i.e. the anchorage and binding of protein to the surface are enthalpy controlled processes whereas the surface denaturation as well as rearrangement and folding is an entropy controlled process. The role of diffusion on rate of adsorption has also been discussed.

Adsorption↗

PubMed1991

Protein adsorption at solid-liquid interfaces: Part IV--Effects of different solid-liquid systems and various neutral salts.

Adsorption isotherms of BSA at the solid-water interfaces have been studied as a function of protein concentration, ionic strength of the medium, pH and temperature using silica, barium sulphate, carbon, alumina, chromium, ion-exchange resins and sephadex as solid interfaces. In most cases, isotherms for adsorption of BSA attained the state of adsorption saturation. In the presence of barium sulphate, carbon and alumina, two types in the isotherms are observed. Adsorption of BSA is affected by change in pH, ionic strength and temperature of the medium. In the presence of metallic chromium, adsorbed BSA molecules are either denatured or negatively adsorbed at the metallic interface. Due to the presence of pores in ion-exchange resins, adsorption of BSA is followed by preferential hydration on resin surfaces in some cases. Sometimes two steps of isotherms are also observed during adsorption of BSA on the solid resins in chloride form. Adsorption of BSA, beta-lactoglobulin, gelatin, myosin and lysozyme is negative on Sephadex surface due to the excess adsorption of water by Sephadex. The negative adsorption is significantly affected in the presence of CaCl2, KSCN, LiCl, Na2SO4, NaI, KCl and urea. The values of absolute amounts of water and protein, simultaneously adsorbed on the surface of different solids, have been evaluated in some cases on critical thermodynamic analysis. The standard free energies (delta G0) of excess positive and negative adsorption of the protein per square meter at the state of monolayer saturation have been calculated using proposed universal scale of thermodynamics. The free energy of adsorption with reference to this state is shown to be strictly comparable to each other. The magnitude of standard free energy of transfer (delta G0B) of one mole of protein or a protein mixture at any type of physiochemical condition and at any type of surface is observed to be 38.5 kJ/mole.

Absorption↗

PubMed1991

Protein adsorption at solid-liquid interfaces: Part III--Adsorption from ternary protein mixture.

Simultaneous adsorption of bovine serum albumin (BSA), beta-lactoglobulin and gelatin from aqueous solutions of their ternary mixture to the alumina-water interface has been studied as a function of protein concentration at different values of pH, ionic strength, temperature and weight fraction ratios of proteins. At a fixed weight fraction of beta-lactoglobulin, preferential adsorption (gamma w(lac)) of this protein significantly depends on the amounts of BSA and gelatin present in the solution before adsorption. At higher ranges of protein concentrations, extent of adsorption (gamma w(ser)) of BSA decreases sharply with increase of gamma w(lac) until gamma w(ser) becomes significantly negative, thereby indicating that beta-lactoglobulin and water preferentially adsorbed at the interface are responsible for complete displacement of BSA from the surface. On the other hand, adsorption (gamma w(gel)) of gelatin under similar situation increases mutually with increase in the values of gamma w(lac) in many systems. In few systems, gamma w(gel) also decreases with increase of gamma w(lac) depending upon solution parameters. At pH 5.2, increase of ionic strength and temperature, respectively, increases the extent of adsorption of each protein in the mixture considerably. Extents of adsorption of all proteins are observed to increase when pH is changed from 5.2 to 6.4. The affinities of different proteins in the mixture are expressed in unified scales either in terms of maximum extents of total adsorption or in terms of standard free energies of adsorption of protein mixtures with respect to surface saturation.

Adsorption↗

PubMed1991

Protein adsorption at solid-liquid interfaces: Part I--Affinities of proteins for alumina surface.

Extent of adsorption (gamma pw) of bovine serum albumin, beta-lactoglobulin, gelatin and myosin at the alumina-water interface has been measured as function of protein concentration (Cp) at several temperatures, pH, and ionic strengths of the medium. gamma pw for proteins in most cases increases with increase of protein concentration but it attains maximum value gamma pw(m) when Cp is high. Values of maximum adsorption have been examined in terms of molecular orientation, molecular size and shape and unfolding of the packed proteins at the interface. In few cases, gamma pw increases with increase of Cp without reaching a real state of saturation as a result of aggregation of molecules or extensive unfolding of the protein at the interface. In the case of beta-lactoglobulin at pH 5.2 and ionic strength 0.05, gamma pw in high concentration region decreases to zero value when Cp increases. For myosin at 45 degrees C and pH 6.4, and also at 27 degrees and pH 7.8, the values of gamma pw are all negative and these negative values increase with increase of Cp. All these results have been explained in terms of significant competitions of water and protein for binding to the surface sites of the powdered alumina. Adsorption of myosin has also been found to be affected in the presence of NaCl, KCl, CaCl2, KI, Na2SO4, LiCl and urea. The relative affinities of the adsorption of various proteins for the surface of alumina at different physical conditions of the system have been compared in terms of maximum values of adsorption attained when gamma pw is varied with Cp. The affinities are shown to be compared more precisely in terms of the standard free energy decrease for the saturation of the surface by protein as a result of the change in its concentration from zero to unity in the mole fraction scale.

Adsorption↗

PubMed1991

Protein adsorption at solid-liquid interfaces: Part II--Adsorption from binary protein mixture.

Extent of adsorption of proteins at alumina-water interface from solutions containing binary mixture of beta-lactoglobulin and bovine serum albumin (BSA), beta-lactoglobulin and gelatin, and gelatin and bovine serum albumin has been estimated as functions of protein concentrations at varying pH, ionic strength, temperature and weight fraction ratios of protein mixture. The extent of adsorption (gamma lacw) of lactoglobulin in the presence of BSA increases with increase of protein concentration (Clac) until it reaches a maximum but a fixed value gamma lacw(m). Extent of adsorption gamma serw also initially increases with increase of protein concentrations until it reaches maximum value gamma serw(m). Beyond these protein concentrations, adsorbed BSA is gradually desorbed due to the preferential adsorption of lactoglobulin from the protein mixture. In many systems, gamma serw at high protein concentrations even becomes negative due to the strong competition of BSA and water for binding to the surface sites in the presence of lactoglobulin. For lactoglobulin-gelatin mixtures, adsorption of both proteins is enhanced as protein concentration is increased until limiting values for adsorption are reached. Beyond the limiting value, lactoglobulin is further accumulated at the interface without limit when protein concentration is high. For gelatin-albumin mixtures, extent of gelatin adsorption increases with increase in the adsorption of BSA. The limit for saturation of adsorption for gelatin is not reached for many systems. At acid pH, adsorbed BSA appears to be desorbed from the surface in the presence of gelatin. From the results thus obtained the role of electrostatic and hydrophobic effects in controlling the adsorption process has been analysed.

Adsorption↗

PubMed1990

Location of a P1 plasmid replication inhibitor determinant within the initiator gene.

The P1 plasmid replication initiator protein, RepA, binds to its own promoter and represses transcription efficiently. There are only about 20 RepA dimers present per repA gene. A possible reason for this highly restrained expression became evident when repA expression was increased by using foreign promoters: with fivefold overexpression, the replication rate was diminished, and with 40-fold overexpression, replication was not detectable. The inhibition was P1 specific: growth of Escherichia coli and replication of pSC101, R6K, and mini-F plasmids were not affected. The activity is apparently not from RepA itself. Excess purified RepA did not inhibit replication in vitro. Mutations of the repA translation initiation codon reduced synthesis of the initiator but not the inhibitory activity. Deletion from either the N- or C-terminal ends of repA (28 and 69 codons, respectively, out of the 286-codon open reading frame) affected the initiator but not the inhibitory activity. Further deletions affected both the activities. These results demonstrate that the integrity of the initiator is not required for inhibition, but involvement of an unstable initiator fragment or of initiator mRNA cannot be ruled out.

Amino Acid Sequence↗

PubMed1989

Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomplete repression of the lytic replicon by the c1 immunity repressor. Incomplete repression was particularly evident in lysogens of the thermoinducible P1 c1.100 prophage, whose replication at 32 degrees C remained almost unaffected when use of the plasmid replicon was prevented. Moreover, the average plasmid copy number of P1 in a P1 c1.100 lysogen was elevated with respect to the copy number of P1 c1+. The capacity of the lytic replicon to act as an auxiliary in plasmid maintenance may contribute to the extraordinary stability of P1 plasmid prophage.

Alleles↗

PubMed1989

Physico-chemical aspects of solubility of myosin in aqueous media.

Solubility of fish (Labio rohita) myosin has been studied at varying temperatures in presence of various inorganic salts like NaCl, KCl, NaBr, Na2SO4, KI, and organic solutes like sucrose and urea. The effect of pH on the solubility has also been studied both in absence and presence of NaCl. Thermal denaturation temperatures of myosin in presence of NaCl, KCl, NaBr and Na2SO4 were found to be 40 degrees, 40 degrees, 45 degrees and 50 degrees C respectively. Thermodynamic parameters like changes in standard free energy (delta G degrees), enthalpy (delta H degrees) and entropy (delta S degrees) for precipitation of myosin from solution phase to gel phase have been evaluated and the physico-chemical aspects have been critically discussed. The average delta G degrees for gel formation varied only between -30 and -40 kJ/mole of myosin, although the nature of solutes, temperature and folding state of protein have been grossly altered. A compensation effect has also been exhibited from the linear plot of average values of delta H degrees against T delta S degrees for various solutes.

Animals↗

PubMed1988

Mini-P1 plasmid replication: the autoregulation-sequestration paradox.

It has been proposed that the initiator protein RepA is rate limiting for mini-P1 plasmid replication, and that the role of the plasmid copy number control locus is to sequester the initiator and thus reduce replication. This proposal appears inconsistent with the observation that RepA is autoregulated, since the protein lost by sequestration should be replenished. A resolution of this autoregulation-sequestration paradox is possible if the sequestered RepA, unavailable for replication, is still available for promoter repression. We demonstrate that RepA binds to the control locus and to the promoter region simultaneously, causing the intervening DNA to loop. DNA looping could provide the requisite mechanism by which RepA bound to the control locus might exert repression.

DNA Helicases↗

PubMed1988

P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites.

The unit-copy plasmid replicon mini-P1 consists of an origin, a gene for an initiator protein, RepA, and a control locus, incA. Both the origin and the incA locus contain repeat sequences that bind RepA. It has been proposed that the incA repeats control replication by sequestering the rate-limiting RepA initiator protein. Here we show that when the concentration of RepA was increased about fourfold beyond its normal physiological level from an inducible source in trans, the copy number of a plasmid carrying the P1 origin increased about eightfold. However, when the origin and a single copy of incA were present in the same plasmid, the copy number did not even double. The failure of an increased supply of RepA to overcome the inhibitory activity of incA is inconsistent with the hypothesis that incA inhibits replications solely by sequestering RepA. We propose that incA, in addition to sequestration, can also restrain replication by causing steric hindrance to the origin function. Our proposal is based on the observation that incA can bind to a RepA-origin complex in vitro.

Bacterial Proteins↗

PubMed1987

Replication of mini-P1 plasmid DNA in vitro requires two initiation proteins, encoded by the repA gene of phage P1 and the dnaA gene of Escherichia coli.

We have developed an in vitro DNA-replication system that replicates exogenously added mini-P1 plasmid DNA. The system consists of purified P1 RepA protein and a partially purified mixture of Escherichia coli replication proteins. It is essentially the same as that described for the replication of oriC plasmid DNA [Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374]. Mini-P1 DNA replication requires the E. coli DnaA initiation protein in addition to the P1 RepA initiation protein. The reaction is inhibited by rifampicin, novobiocin, and antibody to DnaB, suggesting the involvement of RNA polymerase, DNA gyrase, and DnaB protein. Replication is initiated in the region of the P1 origin of replication and proceeds unidirectionally as determined by electron microscopy. Thus, the in vitro system mimics the essential features of mini-P1 replication as suggested by genetic studies.

Bacterial Proteins↗

PubMed1987

P1 plasmid replication: measurement of initiator protein concentration in vivo.

To study the functions of the mini-P1 replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer detectable.

Cloning, Molecular↗

PubMed1986

P1 plasmid replication. Role of initiator titration in copy number control.

The copy number control locus incA of unit copy plasmid P1 maps in a region containing nine 19 base-pair repeats. Previous results from studies in vivo and in vitro indicated that incA interacts with the plasmid-encoded RepA protein, which is essential for replication. It has been proposed that the repeat sequences negatively control copy number by sequestering the RepA protein, which is rate-limiting for replication. Our results lend further support to this hypothesis. Here we show that the repeats can be deleted completely from P1 miniplasmids and the deletion results in an approximately eightfold increase in plasmid copy number. So, incA sequences are totally dispensable for replication and have only a regulatory role. The copy number of incA-deleted plasmids can be reduced if incA sequences are present in trans or are reincorporated at two different positions in the plasmid. This reduction in copy number is not due to lowered expression of the repA gene in the presence of incA. We show that one repeat sequence is sufficient to bind RepA and can reduce the copy number of incA-deleted plasmids. When part of the repeat was deleted, it lost its ability to bind as well as influence copy number. These results show a strong correlation between the capacity of incA repeats to bind RepA protein both in vivo and in vitro, and the function of incA in the control of copy number.

Autoradiography↗

PubMed1985