Effect of Ionic Strength on the Kinetics of Trypsin and Alpha Chymotrypsin

The kinetic effects resulting from changes in the medium ionic strength on reactions involving trypsin or α-chymotrypsin are different. The reaction rate increases continuously as the ionic strength increases with α-chymotrypsin. With trypsin, the rate increases at low ionic strengths but as the ionic strength further increases a gradual inhibitory effect is observed. The effects produced by different salts of various valence types (from uni-univalent to uni-trivalent or tri-univalent) are essentially the same, and they are a function of the square root of the ionic strength. The quantitative differences among the various salts may be accounted for on the basis of individual properties of the ions, such as the size of the hydrated ion, "association," etc. The effects of salts on the enzymic reactions described herein are amenable to the same electrostatic treatment applicable to non-enzymatic reactions. By applying Brönsted's basic kinetic concepts and the Debye-Hückel law of electrolyte activity, it appears that the salt effects are mainly due to changes in the dissociation of ionizable groups. This appears to be a general method for analyzing the effect of inorganic ions on enzymic reactions.     

Effect of the Medium Dielectric Strength on the Activity of Alpha Chymotrypsin

The rates of hydrolysis of TrEE, TEE, and ATEE¹ by α-chymotrypsin were determined in media of variable dielectric strength. Many substances which modify the dielectric constant of the medium, exert additional specific effects on the reaction rate, noticeable at more or less elevated concentrations. Notwithstanding, it is possible to differentiate the dielectric and specific effects by comparing the rates in solvents of distinct nature at relatively low concentrations. Thus, the effect of varying the dielectric strength could be studied within wider ranges (ΔD = 20 with TrEE and ca. 28 with ATEE) than in the previous study of trypsin (ΔD = 12). The dielectric effect on α-chymotrypsin is the opposite of that observed with trypsin. In both cases there is a linear relationship between the logarithm of the rate of hydrolysis and the reciprocal of the dielectric constant. The slope is negative with α-chymotrypsin and positive with trypsin. According to expressions relating the dielectric constant to the rate in non-enzymatic reactions, the behavior of α-chymotrypsin is like that of a negative ion, while trypsin behaves as a positive ion. The enzyme activity appears to depend upon the arrangement of charges in the enzyme and substrate molecules, rather than on the presence of certain atomic groupings in the substrate.     

The Effect of Proteolytic Enzymes on E. coli Phages and on Native Proteins

Lambda coli phage is not inactivated by chymotrypsin, trypsin, or ficin. T₂ phage is slowly inactivated by high concentrations of (α-, β-, γ-, or Δ-chymotrypsin, but not by trypsin or ficin. P₁ phage is slowly inactivated by α-, β-, or γ-chymotrypsin, or ficin, more rapidly by Δ-chymotrypsin, and much more rapidly by trypsin. Crystalline egg albumin, crystalline serum albumin, E. coli nucleoprotein, and yeast nucleoprotein are hydrolyzed slowly by α-chymotrypsin. Yeast nucleoprotein, like P₁ phage, is hydrolyzed more rapidly by Δ-chymotrypsin than by α-chymotrypsin, but not by trypsin or ficin. Neither phages nor native proteins were attacked by papain, carboxypeptidase, deoxyribonuclease, or ribonuclease.     

The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

The autolysis of trypsin and α-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in `inert' vessels) are second-order for both trypsin and α -chymotrypsin, with rate constants of 13.0mol<sup>−1</sup>·dm<sup>3</sup>·s<sup>−1</sup> for trypsin (in 50mm-NaCl at pH8.0 at 25°C) and 10.2mol<sup>−1</sup>·dm<sup>3</sup>·s<sup>−1</sup> for α-chymotrypsin (in 0.1m-glycine at pH9.2 at 30°C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and α-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of α-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules is to shift the pH–activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the `microenvironment' of the enzyme associated with the negatively charged surface or macromolecule.     

Affinity chromatography of bovine trypsin. A rapid separation of bovine α- and β-trypsin

Affinity adsorbents for bovine trypsin were prepared by covalently coupling p-(p′-amino-phenoxypropoxy)benzamidine to cellulose and to agarose. Trypsin binds to both adsorbents at pH6–8 and is released at low pH values or in the presence of n-butylamine hydrochloride. Pure β-trypsin may be eluted from crude trypsin bound at pH8.0 to the cellulose adsorbent by stepwise elution with an acetate buffer, pH5.0. Both α- and β-trypsin may be isolated by chromatography of crude trypsin on the agarose derivative in an acetate buffer, pH4.0. These two methods for purifying the trypsin are specific to the particular adsorbents. They are rapid and convenient in use. Both methods leave a mixture of the two enzymes bound to the adsorbent and release occurs only at low pH values. The effects of pH, composition and ionic strength of buffer and other variables on both purification methods are described. Affinity adsorbents of soya-bean trypsin inhibitor and of N-α-(N′-methyl-N′-sulphanilyl) sulphanilylagmatine bound to agarose were prepared, but were found to be of limited usefulness in the purification of trypsin.     

Production of an Amylase-Sensitive Bacteriocin by an Atypical Leuconostoc paramesenteroides Strain

An atypical Leuconostoc paramesenteroides strain isolated from retail lamb produced a bacteriocin, leuconocin S, that was inactivated by α-amylase, trypsin, α-chymotrypsin, protease, and proteinase K but not by lipase or heat treatment at 60°C for 30 min. Supernatants from culture broths produced two glycoprotein bands on sodium dodecyl sulfate-polyacrylamide gels; these had molecular weights of 2,000 and 10,000 and activity against Lactobacillus sake ATCC 15521. The crude bacteriocin preparation was bacteriostatic and dissipated proton motive force. Bacteriocin activity was produced over a wide pH range (5.2 to 7.9) on buffered agar medium, with an optimum pH of pH 6.15. The optimum pH for production in broth was 6.5 to 7.0.     

CHEMICAL ANTAGONISM OF IONS : IV. EFFECT OF SALT MIXTURES ON GLYCINE ACTIVITY.

The pH of a 0.01 molar solution of glycine, half neutralized with NaOH, is 9.685. Addition of only one of the salts NaCl, KCl, MgCl₂, or CaCl₂ will lower the pH of the solution (at least up to 1 µ). If a given amount of KCl is added to a glycine solution, the subsequent addition of increasing amounts of NaCl will first raise the pH (up to 0.007 M NaCl). Further addition of NaCl (up to 0.035 M NaCl) will lower the pH, and further additions slightly raise the pH. The same type of curve is obtained by adding NaCl to glycine solution containing MgCl₂ or CaCl₂ except that the first and second breaks occur at 0.015 M and 0.085 M NaCl, respectively. Addition of CaCl₂ to a glycine solution containing MgCl₂ gives the same phenomena with breaks at 0.005 M and 0.025 M CaCl; or at ionic strengths of 0.015 µCaCl₂ and 0.075 µCaCl₂. This indicates that the effect is a function of the ionic strength of the added salt. These effects are sharp and unmistakable. They are almost identical with the effects produced by the same salt mixtures on the pH of gelatin solutions. They are very suggestive of physiological antagonisms, and at the same time cannot be attributed to colloidal phenomena.     

Globulin-specific Proteolytic Activity in Germinating Pumpkin Seeds as Detected by a Fluorescence Assay Method

The proteolytic activities of α-chymotrypsin, trypsin, pepsin, bromelain, and an extract from germinating pumpkin seeds (Cucurbita moschata) were determined by their ability to effect the release of 1-anilino-8-naphthalenesulfonate bound to internal hydrophobic sites in intact protein substrates. Casein, glyceraldehyde-3-P dehydrogenase, urease, catalase, pumpkin seed globulin, and bovine serum albumin enhanced the fluorescence of 1-anilino-8-naphthalenesulfonate sufficiently to be used as proteolytic substrates. Chymotrypsin, trypsin, pepsin, and bromelain exhibited activity against all or almost all of the protein substrates. The activity of 1 μg of α-chymotrypsin or trypsin and 100 ng of pepsin could be easily detected by this method of assay within 4 to 5 minutes depending upon the substrate. The enzyme extracted from 3-day germinated pumpkin seeds exhibited strong activity only against pumpkin seed globulin, weak activity against the globulins of squash and cucumber and casein, and no activity against the other protein substrates. Activity against pumpkin globulin was maximal at pH 7.4. When assayed by an increase in ninhydrin-positive products, the enzyme extract from pumpkin seeds also showed strong activity against pumpkin globulin and weak activity against casein. The 1-anilino-8-naphthalenesulfonate-fluorescence method was at least 20 times more sensitive than the ninhydrin method and was 10 to 20 times more rapid.     

Stability of Secondary and Tertiary Structures of Virus-Like Particles Representing Noroviruses: Effects of pH,Ionic Strength,and Temperature and Implications for Adhesion to Surfaces

Loss of ordered molecular structure in proteins is known to increase their adhesion to surfaces. The aim of this work was to study the stability of norovirus secondary and tertiary structures and its implications for viral adhesion to fresh foods and agrifood surfaces. The pH, ionic strength, and temperature conditions studied correspond to those prevalent in the principal vehicles of viral transmission (vomit and feces) and in the food processing and handling environment (pasteurization and refrigeration). The structures of virus-like particles representing GI.1, GII.4, and feline calicivirus (FCV) were studied using circular dichroism and intrinsic UV fluorescence. The particles were remarkably stable under most of the conditions. However, heating to 65°C caused losses of β-strand structure, notably in GI.1 and FCV, while at 75°C the α-helix content of GII.4 and FCV decreased and tertiary structures unfolded in all three cases. Combining temperature with pH or ionic strength caused variable losses of structure depending on the particle type. Regardless of pH, heating to pasteurization temperatures or higher would be required to increase GII.4 and FCV adhesion, while either low or high temperatures would favor GI.1 adhesion. Regardless of temperature, increased ionic strength would increase GII.4 adhesion but would decrease GI.1 adhesion. FCV adsorption would be greater at refrigeration, pasteurization, or high temperature combined with a low salt concentration or at a higher NaCl concentration regardless of temperature. Norovirus adhesion mediated by hydrophobic interaction may depend on hydrophobic residues normally exposed on the capsid surface at pH 3, pH 8, physiological ionic strength, and low temperature, while at pasteurization temperatures it may rely more on buried hydrophobic residues exposed upon structural rearrangement.     

Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Amide proton NMR signals from the N-terminal domain of monomeric α-synuclein (αS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in α-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of αS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Cα chemical shift data point to <10% residual α-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of αS encapsulated within living Escherichia coli cells. Our finding that temperature effects on αS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that αS remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of αS with membranes and fibrils.     

1H n.m.r. of the DNA-dependent RNA polymerase from Escherichia coli

The ¹H n.m.r. study of the DNA-dependent RNA polymerase from Escherichia coli has revealed that the holoenzyme (ββ′α₂σ) displays two mobile regions: one, observable also in the core enzyme (ββ′α₂), is characterized by basic amino acids and its appearance and form depend on ionic strength; the other, specific to the holoenzyme, is characterized by threonine residues and its appearance does not depend on ionic strength.     

Effect of method of preparation on the States of chlorophyll in euglena chloroplast fragments as determined by fluorescence spectroscopy

The state of chlorophyll in chloroplast fragments is affected by such factors as the ionic strength and pH of the suspending medium. With increasing ionic strength or at pH values other than neutrality, there is a decrease in the fluorescence yield of the form of chlorophyll with fluorescence maximum at 715 to 736 mμ (aggregate) and an increase in the yield of the form with fluorescence maximum at 685 mμ (monomer). (Positions of maxima cited are for 77°K.) These changes in yield are accompanied by modifications in absorption and fluorescence excitation spectra. It is also noted that these effects are similar to the ones brought about by pancreatic lipase, wheat germ lipase, pancreatic trypsin or urea. An interpretation is given which is consistent with the experimental data, namely, that the effects originate in conformational changes in the proteins to which the pigments are attached. These conformational changes give rise to an increase in the size of the aggregate and a decrease in the probability of energy transfer between the monomer and aggregates.     

Electrodichroism of Purple Membrane: Ionic Strength Dependence

The dichroism of purple membrane suspension was measured in dc and ac electric fields. From these measurements three parameters can be obtained: the permanent dipole moment, μ, the electrical polarizability, α, and the retinal angle, δ, (relative to the membrane normal). The functional dependence of the dichroism on the electric field is analyzed. There is a small decrease (~2°) in retinal angle going from dark adapted to the light adapted form. No measurable difference in μ, α, and δ was found under the photocycle. The dichroism was measured in two different salt solutions (KCl and CaCl₂) in the range 0-10 mM. The retinal angle increases from 64° to 68° with increasing ionic strength going through a minimum. This is attributed to the changing (decreasing) inner electric field in the membrane. The polarizability, α, consists of two parts. One component is related to the polarization of the purple membrane and the second component to the ionic cloud. The second component decreases with ion concentration approximately as κ^-3 (κ is the Debye parameter) in agreement with a model calculation for the polarization of the ionic cloud. The origin of the slightly ionic strength dependent permanent dipole moment is not well understood.     

Roles of the Src Tyrosine Kinases Lck and Fyn in Regulating γδTCR Signal Strength

Lck and Fyn, members of the Src family of tyrosine kinases, are key components of the αβTCR-coupled signaling pathway. While it is generally accepted that both Lck and Fyn positively regulate signal transduction by the αβTCR, recent studies have shown that Lck and Fyn have distinct functions in this signaling pathway, with Lck being a positive regulator and Fyn being a negative regulator of αβTCR signal transduction. To determine whether Lck and Fyn also differentially regulate γδTCR signal transduction, we analyzed γδ T cell development and function in mice with reduced Lck or Fyn expression levels. We found that reducing Lck or Fyn levels altered the strength of the γδTCR signaling response, with low levels of Lck weakening γδTCR signal strength and low levels of Fyn augmenting γδTCR signal strength. These alterations in γδTCR signal strength had profound effects not only on αβ/γδ lineage choice, but also on γδ thymocyte maturation and γδ T cell effector function. These results indicate that the cellular levels of Lck and Fyn play a role in regulating the strength of the γδTCR signaling response at different stages in the life of the γδ T cell.     

Immunochemical Studies on Arachis hypogaea Proteins With Particular Reference to the Reserve Proteins. I. Characterization, Distribution, and Properties of alpha-Arachin and alpha-Conarachin

Fourteen antigenic constituents have been detected in Arachis hypogaea seeds. The major proteins of the classic arachin and conarachin fractions have been identified. Arachin contains 4 antigens (the major one called α-arachin) and conarachin contains 2 which have been called α₁, and α₂-conarachin. Structural differences between α-arachin, α₁ and α₂-conarachin are indicated by their different antigenic specificities. α-Arachin precipitates as a separate entity at low temperature. The action of trypsin on this protein induces an increase in electrophoretic mobility and prevents precipitation at low temperature. This enzyme has no detectable effect on α₁ and α₂-conarachin.     

CRYSTALLINE TRYPSIN : IV. REVERSIBILITY OF THE INACTIVATION AND DENATURATION OF TRYPSIN BY HEAT

1. If dilute solutions of purified trypsin of low salt concentration at pH from 1 to 7 are heated to 100°C. for 1 to 5 minutes and then cooled to 20°C. there is no loss of activity or formation of denatured protein. If the hot trypsin solution is added directly to cold salt solution, on the other hand, all the protein precipitates and the supernatant solution is inactive. 2. The per cent of the total protein and activity present in the soluble form decreases from 100 per cent to zero as the temperature is raised from 20°C. to 60°C. and increases again from zero to 100 per cent as the solution is cooled from 60°C. to 20°C. The per cent of the total protein present in the soluble (native) form at any one temperature is nearly the same whether the temperature is reached from above or below. 3. If trypsin solutions at pH 7 are heated for increasing lengths of time at various temperatures and analyzed for total activity and total protein nitrogen after cooling, and for soluble activity and soluble (native) protein nitrogen, it is found that the soluble activity and soluble protein nitrogen decrease more and more rapidly as the temperature is raised, in agreement with the usual effects of temperature on the denaturation of protein. The total protein and total activity, on the other hand, decrease more and more rapidly up to about 70°C. but as the temperature is raised above this there is less rapid change in the total protein or total activity and at 92°C. the solutions are much more stable than at 42°C. 4. Casein and peptone are not digested by trypsin at 100°C. but when this digestion mixture is cooled to 35°C. rapid digestion occurs. A solution of trypsin at 100°C. added to peptone solution at zero degree digests the peptone much less rapidly than it does if the trypsin solution is allowed to cool slowly before adding it to the peptone solution. 5. The precipitate of insoluble protein obtained from adding hot trypsin solutions to cold salt solutions contains the S-S groups in free form as is usual for denatured protein. 6. The results show that there is an equilibrium between native and denatured trypsin protein the extent of which is determined by the temperature. Above 60°C. the protein is in the denatured and inactive form and below 20°C. it is in the native and active form. The equilibrium is attained rapidly. The results also show that the formation of denatured protein is proportional to the loss in activity and that the re-formation of native protein is proportional to the recovery of activity of the enzyme. This is strong evidence for the conclusion that the proteolytic activity of the preparation is a property of the native protein molecule.     

Isolation and properties of highly purified Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polumerase

1. The subunits α and β of Halobacterium cutirubrum DNA-dependent RNA polymerase have been purified to electrophoretic homogeneity. Both have mol.wt. 18000 and they are required in equimolar amounts for optimum activity. 2. The instability of the complete enzyme, αβ, in the absence of salt is due to the rapid inactivation of the β subunit in these conditions. 3. Nearest-neighbour analysis of the product formed on poly[d(A-T)] as template shows that the enzyme copies the latter accurately. 4. The enzyme initiates new chains with purine nucleoside triphosphates exclusively. 5. The product obtained in the standard assay conditions contains some high mol.wt. (>16S) material, but consists primarily of short chains, of average length 70–80 nucleotide units. 6. The template specificity of the complete enzyme has been studied at high and low ionic strength. Its extreme dependence on salt concentration is unrelated to the gross overall base composition of the DNA used. 7. T₇ DNA is transcribed asymmetrically and the enzyme selectively copies the T₇ `early' genes. 8. Preliminary amino acid analyses of α and β subunits show that their overall content of acidic, basic and neutral amino acids does not differ appreciably from that of Escherichia coli RNA polymerase.     

A conformation change of single stranded polyriboadenylate induced by an electric field

Absorbance measurements performed with high molecular weight poly A at pH 8 show that the degree of single strand stacking present at high ionic strength is reduced at low ionic strengths. The salt dependence of the poly A conformation is assigned to an electrostatic repulsion between subsequent turns of the single strand “helix” structure. - Electric fields of 5 to 80 kV/cm induce an increase in the poly A absorbance consistent with a decrease in the ion concentration in the environment of the polymer. The increase of the absorbance is a linear function of the field strength suggesting that the conformation change is caused by a dissociation field effect. At increasing ionic strength, threshold values of the electric field strength have to be exceeded in order to induce measurable absorbance changes. - The time required for the conformation change decreases from about 2 μsec at 10⁻⁴ M ionic strength to about 0.3 μsec at high ionic strengths. At low ionic strengths the ion equilibration may influence the rate limiting step, whereas the arrangement of the nucleotide residues into the ordered structure is rate limiting at high ionic strengths.     

Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids

The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF₆] at 80 °C.     

The action of alpha chymotrypsin upon mixtures of esters and proteins at enzyme-saturating concentrations

The behavior of α-chymotrypsin has been studied in the simultaneous presence of two different substrates, each present in the reaction mixture at its saturation level. Mixtures of two esters were hydrolyzed at rates intermediate between the rates of hydrolysis of each ester when present alone, suggesting, in this case, competitive hydrolysis. In contrast, the rates of hydrolysis in mixtures of casein with gelatin or of either protein with an ester were equal to the sum of the rates of hydrolysis of the separate substrates, indicating in these cases independent hydrolysis. The activity of the α-chymotrypsin preparation used could not be attributed to contamination with other enzymes. Studies of the effect of soy bean inhibitor on chymotrypsin indicate that the mechanism of inhibition with protein substrates differs from that when esters are used, providing further evidence that α-chymotrypsin reacts differently with esters and proteins. These results indicate that if chymotrypsin forms specific complexes with its substrates, it must possess at least three distinct active sites. However there is independent chemical evidence that the proteolytic and esterolytic activities of this enzyme reside in the same active center. If this is true, the experimental observations reported here cannot be explained unless it is supposed that this enzyme does not form specific Michaelis complexes with its substrates.