Polymerization studies on protein from the dahlemense strain of tobacco mosaic virus: Light scattering and related studies

Light-scattering and related studies on protein of Dahlmense strain of tobacco mosaic virus (DTMV) show that its polymerization characteristics are considerably different from those of TMV protein. At pH 6.0 in phosphate buffer (I = 0.1), the extent of polymerization of DTMV protein is greater than that of TMV protein, they are nearly the same at pH 6.25, and that of DTMV protein is less than that of TMV protein at pH 6.5. At pH 7.0 and 7.5, DTMV protein polymerizes more readily than TMV protein. Similar studies in phosphate buffer (I = 0.05) show that the extent of polymerization for DTMV protein is less than that of TMV protein at pH 6.0 and almost negligible at pH 6.25. Acid-base titration studies show that, upon temperature-mediated polymerization, about 2 H⁺ ions are bound per monomer of DTMV protein at pH 6.O.Electron microscope studies show that DTMV protein exists at room temperature as double discs and polymerized rods in phosphate buffer at pH 7.5, I = 0.1; at pH values below 6.5, DTMV protein is entirely in the form of polymerized rods. Velocity sedimentation studies of DTMV protein at room temperature are in agreement with these findings. At low temperatures, except at pH 7.5, most of the material sedimented with an s value of around 25 S. Thus, at low temperatures, except at pH 7.5, DTMV protein in solution is in the form of particles the size of double discs with an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 596,000 g/mole or even larger. Therefore, temperature-mediated polymerization of DTMV protein at pH values below 6.5 in phosphate buffer (I = 0.1) and below 6.25 in phosphate buffer (I = 0.05) involves particles at least as large as double discs as the starting material.     

The Donnan effect in tobacco mosaic virus and its components

Osmotic pressure studies were carried on tobacco mosaic virus (TMV) and its components, protein and RNA, as well as on bis(3,3′-aminopropyl)amine, reported to be present in TMV preparations. Solvents were phosphate and barbital buffers at different values of pH and ionic strength. Measurements were made at room temperature. The Donnan effect was exhibited by TMV protein in phosphate buffer of 0.01 ionic strength at pH values ranging between 5.8 and 7.5. The observed values of the Donnan effect at pH 5.8 and 5.97 were in reasonable agreement with theoretical values calculated from the charge obtained by hydrogen ion titration. TMV-RNA in phosphate buffer at pH 7.5 and ionic strength 0.01 did not exhibit more than 1% of the expected Donnan effect. This is explained tentatively as the result of firm binding of metal ions. Negative values of osmotic pressure were observed with bis(3,3′-aminopropyl)amine. Similar anomalous osmosis was sometimes observed with TMV protein and with TMV. In agreement with earlier observations, TMV did not exhibit the Donnan effect in phosphate buffer of 0.01 ionic strength at pH values ranging from 5.5 to 8.0. However, TMV dialysed extensively in the presence of EDTA at pH 8.5 and TMV produced by reconstitution of purified protein and RNA did exhibit the Donnan effect in both phosphate and barbital buffers. The magnitude was of the same order as that calculated from the net charge determined by hydrogen ion titration. When reconstituted TMV, which did exhibit Donnan effect, was treated with calcium ions, the effect was abolished.     

Effect of dipolar ions on the entropy-driven polymerization of tobacco mosaic virus protein

The effect of the dipolar ions, glycine, glycylglycine, and glycylglycylglycine on the polymerization of tobacco mosaic virus (TMV) protein has been studied by the methods of light scattering and ultracentrifugation. All three dipolar ions promote polymerization. The major reaction in the early stage is transition from the 4 S to the 20 S state. As in the absence of dipolar ions, the polymerization is enhanced by an increase in temperature; it is endothermic and therefore entropy-driven. The effect of the dipolar ions can be understood in terms of their action as salting-out agents; they increase the activity coefficient of TMV A protein, the 4 S material, and thus shift the equilibrium toward the 20 S state. The salting-out constants, K, for the reaction in 0.10 ionic strength phosphate buffer at pH 6.7 was found by the light scattering method to be 1.6 for glycine, 2.5 for glycylglycine, and 2.5 for glycylglycylglycine. A value of 2.7 was obtained by the ultracentrifugation method for glycylglycine in phosphate buffer at 0.1 ionic strength and pH 6.8 at 10 degrees C. For both glycine and glycylglycine, K increases when the ionic strength of the phosphate buffer is decreased. This result suggests that electrolytes decrease the activity coefficient of the dipolar ions, a salting-in phenomenon. However, the salting-in constants evaluated from these results are substantially higher than those previously determined by solubility measurements. The effect of glycine and glycylglycine on polymerization was studied at pH values between 6.2 and 6.8. The effectiveness of both dipolar ions is approximately 50% greater at pH 6.8 than at pH 6.2. The variation of the extent of polymerization with pH in the presence of the dipolar ions is consistent with the interpretation that approximately one hydrogen ion is bound for half of the polypeptide units in the polymerized A protein.     

Sedimentation equilibrium measurements of the intermediate-size tobacco mosaic virus protein polymers

Short-column sedimentation equilibrium methods have been applied for the first time to tobacco mosaic virus (TMV) protein (0.1 M ionic strength orthophosphate) at pH 6.5 and at pH 7.0 to estimate molecular weights. Previous sedimentation velocity experiments at pH 6.5, 20 degrees C have led to the conclusion that the major boundary with an S0(20),w value of 24.4 +/- 0.1 S consists of a distribution of polymers which are mainly three-turn, 48-51-subunit helical rod aggregates. The directly measured z-average molecular weights together with sedimentation velocity data are entirely consistent with this assignment of a three-turn aggregate. Molecular weights have also been determined under two conditions where a large mass fraction of the protein sediments with an S0(20),w value of 20.3 +/- 0.2 S. At pH 6.5, 6-8 degrees C, the aggregates in this boundary are metastable and correspond to 50-60% of the preparation. At pH 7.0, 20 degrees C at equilibrium, 65-75% of the protein sediments at 20.3 S. The 20.3S boundary is very similar under both conditions and is interpreted as being composed of a distribution of protein aggregates centered about 39 +/- 2 subunits. This result is important in the interpretation of previous kinetic measurements of TMV self-assembly. The current view is that the 34-subunit structure of TMV protein, in the form of a cylindrical disk which is made up of two 17-subunit layers and has been characterized in single-crystal X-ray diffraction studies, plays a central role in the initial binding steps with RNA. The present results are not consistent with the view that there is a significant concentration of the TMV protein disk structure in solution under the usual conditions of TMV self-assembly.     

Disk aggregates of tobacco mosaic virus protein in solution: electron microscopy observations

Previous studies of the coat protein of tobacco mosaic virus (TMVP) have shown that TMVP presumably exists as linear stacks of two-ring cylindrical disks in the 0.7 M ionic strength buffer used for crystallizing the disks for X-ray diffraction studies [Raghavendra, K., Adams, M.L., & Schuster, T.M. (1985) Biochemistry 24, 3298-3304]. The spectroscopic and sedimentation studies of solutions of TMVP under these crystallizing conditions have demonstrated a long-term metastability of these disk aggregates when they are placed in 0.1 M ionic strength buffers, as are used for reconstituting tobacco mosaic virus from TMVP and viral RNA. The present work describes an electron microscopic study of TMVP disk aggregates under the same solution conditions employed in the previous spectroscopic and sedimentation studies. The results show that in the pH 8.0 0.7 M ionic strength crystallization buffer TMVP exists as stacks of disks which range in size from about 6 to 24 layers, corresponding to 3-12 2-layer disk aggregates having 17 subunits per layer. These TMVP aggregates persist in a metastable form in 0.1 M ionic strength virus reconstitution buffer with no apparent changes in structure of the stacked disks. The results are consistent with the conclusions of the solution physical-chemical studies which suggest that the disk structure may not be related to the 20S TMVP aggregate that is the nucleation species in virus     

Apparent oxidation-reduction potential of Clostridium acidi-urici ferredoxin. Effect of pH, ionic strength, and amino acid replacements.

The effects of pH and ionic strength on the midpoint reduction potential (Emp) of Clostridium acidi-urici ferredoxin were determined using hydrogen gas and hydrogenase. The Emp of native ferredoxin at 24-25 degrees in 0.1 M Tris-chloride buffer, pH 7.0, is--0.434 V. In the pH range examined, the Emp becomes approximately 13 mv more negative per each pH unit increase. A plot of the log of ionic strength versus the apparent Emp of ferredoxin in 0.1 M Tris-chloride buffer, pH 7.5, Was linear over the range of 1.0 to 0.01 ionic strength with Emp values of--0.414 and--0.475 V, respectively, at these extremes. This effect is the same with sodium chloride, sodium bromide, or ammonium sulfate. Potassium phosphate buffer caused a similar change, but the absolute values of Emp differed from those obtained in the presence of the other salts. This effect of pH and ionic strength on Emp may be general for clostridial-type (Fe4S4)2-ferredoxins, since the apparent Emp of Clostridium pasteurianum ferredoxin is affected in a similar manner by these two variables. The Emp of this ferredoxin in 0.1 M Tris-chloride buffer pH 7.0, is--0.405 V. Since the NH2-terminal amino acid residue, Ala1, and Tyr2 of C. acidi urici ferredoxin are near an (Fe4S4)2-cluster in the protein, the apparent Emp of derivatives that contained amino acid replacements in these two positions were determined. Under similar conditions, the Emp of most of the 13 derivatives examined, including those of [Leu2]- and[3-NH2-Tyr30]ferredoxin, is approximately the same as that of native ferredoxin. However, the Emp of [His2]ferredoxin is approximately 15 mv more positive, whereas that of [Trp2]ferredoxin is 22 mv more negative than that of native C. acidi-urici ferredoxin. Variations in sodium chloride concentration and pH also affected the apparent Emp of the derivatives. It is suggested that the changes observed in the Emp of C. acidi-urici ferredoxin are caused by protein conformational changes.     

Sedimentation equilibrium studies of human chymotrypsin II and bovine -chymotrypsin

Sedimentation equilibrium experiments indicate that neither human chymotrypsin II nor bovine δ-chymotrypsin molecules undergo association in the pH range 3–5 where dimerization occurs with α-chymotrypsin. The weight-average molecular weights of human chymotrypsin II and δ-chymotrypsin in a pH 4.4 0.1 ionic strength buffer are 26,200 and 26,400, respectively, using the measured partial specific volumes of 0.722 and 0.727 ml/g at 25 °C. Number-average molecular weight calculations also support the presence of monomeric species at this pH. In the pH range 6–7.6 where sedimentation velocity studies have shown that δ-chymotrypsin associates at concentrations above 3 mg/ml, no association was observed for either the human chymotrypsin II or bovine δ-chymotrypsin in the sedimentation equilibrium experiments where protein concentrations were below 1.2 mg/ml. These studies provide additional evidence that human chymotrypsin II is similar to bovine δ-chymotrypsin.     

Alfalfa mosaic virus protein polymerization.

The self-association of alfalfa mosaic virus coat protein was studied by sedimentation analysis and electron microscopy under a wide range of conditions. In the depolymerized state the protein exists as a molecular species with a sedimentation constant of roughly 3 S and with a molecular weight of (48.4 ± 1.1) × 10³. This value is, within experimental error, twice the value of the monomer (van Beynum, 1975). The dimer has a very stable configuration, as no evidence was found for a monomer-dimer equilibrium between pH values of 3 and 9 and values of ionic strength up to 1.0. One main type of association product (30 S) was found with a molecular weight of (1.48 ± 0.03) × 10⁶. Therefore this particle accomodates 30 dimers which are arranged according to a point group symmetry of 532. The orientation of the 30 dimers within the icosahedral lattice must be such that lattice dyads coincide with the 2-fold axes of the dimers. Micrographs of the 30 S particles show a diameter of about 123 Å; analysis of linear arrays of these particles shows that at low resolution the particle is a hollow sphere with an average coat thickness of about 40 Å.The influence of pH, ionic strength, protein concentration and the type of buffer on the polymerization was determined to some extent and is discussed. The assembly of dimers into the icosahedral particle is an entropy-driven process (Lauffer, 1975); this is concluded from studying the temperature-dependence of the free energy change. Under favourable conditions (phosphate buffer pH 5.5 and ionic strength 0.5) the average enthalpy and entropy changes for the insertion of one dimer into the lattice are about 6.4 kilocalories per mole and 50 entropy units, respectively, based on the unit mole fraction.     

Interaction of tobacco mosaic virus and tobacco mosaic virus protein with bovine serum albumin.

Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.     

Biophysical studies on the lipid transfer particle from the hemolymph of the tobacco hornworm, Manduca sexta

Hydrodynamic studies conducted in the analytical ultracentrifuge provided evidence for two populations of lipid transfer particle (LTP) when centrifuged in a buffer solution containing 10 mM Tris, pH 8.0/100 mM KCl. The apparent sedimentation coefficients of the two species was 23.3 S and 15.3 S. Upon changing the buffer pH to 7.0 or 5.7, two species of LTP were still present but the ratio of their relative abundance was altered. When the KCl concentration in the buffer was lowered to 50 mM the sample sedimented as a single species with an apparent S20,w of 22.9 S. In higher ionic strength buffers (10 mM succinate, pH 5.7/500 mM KCl) LTP sedimented with an apparent S20,w of 14.8 S. Further experiments revealed that these two forms are interconvertable as a function of buffer ionic strength. Given previous estimates of the molecular size of LTP we concluded that the slower sedimenting peak observed at high ionic strength represents monomeric LTP while the faster sedimenting material observed at low ionic strength is likely to be an aggregated state of LTP. This interpretation is supported by molecular weight determinations made by sedimentation equilibrium experiments conducted in 10 mM succinate, pH 5.7/500 mM KCl which yielded a particle Mr = 887,000. Circular dichroism spectra of monomeric LTP sample revealed 6% alpha-helix, 49% beta-sheet, 7% beta-turn and 35% random coil while aggregated LTP contained 13% alpha-helix, 66% beta-sheet and 21% random coil. The transfer activity of the two LTP forms was assayed and found to be the same indicating that either the state of LTP aggregation did not affect transfer activity or that upon exposure to a large excess of lipoprotein substrate disaggregation, without loss of activity, occurs.     

A mechanism of macroscopic (amorphous) aggregation of the tobacco mosaic virus coat protein

To gain more insight into the mechanisms of heating-induced irreversible macroscopic aggregation of the tobacco mosaic virus (TMV) coat protein (CP), the effects of pH and ionic strength on this process were studied using turbidimetry, CD spectroscopy, and fluorescence spectroscopy. At 42 degrees C, the TMV CP passed very rapidly (in less than 15s) into a slightly unfolded conformation, presumably because heating disordered a segment of the subunit where the so-called hydrophobic girdle of the molecule resides. We suppose that the amino acid residues of this girdle are responsible for the aberrant hydrophobic interactions between subunits that initiate macroscopic protein aggregation. Its rate increased by several thousands of times as the phosphate buffer molarity was varied from 20 to 70 mM, suggesting that neutralization of strong repulsive electrostatic interactions of TMV CP molecules at high ionic strengths is a prerequisite for amorphous aggregation of this protein.     

Tobacco mosaic virus protein: sedimentation equilibrium studies of the initial stages of polymerization.

The lowest stages of polymerization of tobacco mosaic virus protein were studied by means of high-speed sedimentation equilibrium experiments. Several distinct modes of polymerization were found. At pH 7.1 the expected monomer-trimer-higher polymer equilibrium was observed--very little dimer was detected at this pH. At pH 7.5, however, a strong dimerization was observed--neither monomer nor trimer was detected at this pH. An octamer appeared to be the only species present other than the dimer. When 0.01 M beta-mercaptoethanol was added to the solvent pH 7.5, the dimer was dissociated, resulting in a monomer-trimer association. The dimerization may be the basis for the larger "doubled" polymers formed by the protein at alkaline pH, while the octamer may correspond to the 8S peak frequently observed in sedimentation velocity experiments at alkaline pH. On the other hand, the monomer-trimer-higher polymer equilibrium may correspond to the single helix formed by the protein at slightly acid pH and to the combination of 4S and 20S peaks seen in sedimentation velocity experiments at slightly acid pH.     

Studies on the mechanism of assembly of tobacco mosaic virus.

Sedimentation and proton binding studies on the endothermic self-association of tobacco mosaic virus (TMV) protein indicate that the so-called "20S" sedimenting protein is an interaction system involving at least the 34-subunit two-turn yield cylindrical disk aggregate and the 49-subunit three-turn helical rod. The pH dependence of this overall equilibrium suggests that disk formation is proton-linked through the binding of protons to the two-turn helix which is not present as significant concentrations near pH 7. There is a temperature-induced intramolecular conformation change in the protein leading to a difference spectrum which is complete in 5 x 10(-6) s at pH 7 and 20 degrees C and is dominated at 300 nm by tryptophan residues. Kinetics measurements of protein polymerization, from 10(-6) to 10(3) s, reveal three relaxation processes at pH 7.0, 20 degrees C, 0.10 M ionic strength K (H) PO4. The fastest relaxation time is a few milliseconds and represents reactions within the 4S protein distribution. The second fastest relaxation is 50-100 x 10(-3) s and represents elementary polymerization steps involved in the formation of the approximately 20 S protein. Analysis of the slowest relaxation, approximately 5 x 10(4) s, suggests that this very slow formation of approximately 20 S protein may be dominated by some first order process in the overall dissociation of approximately 20S protein. Sedimentation measurements of the rate of TMV reconstitution, under the same conditions, show by direct measurements of 4S and approximately 20S incorporation at various 4S to approximately 20S weight ratios that the relative rate of approximately 20S incorporation decreases almost linearly, from 0 to 50% 4S. There appears to be one or more regions of TMV-RNA, approximately 1-1.5 kilobases long, which incorporates approximately 20S protein exclusively. Solutions of approximately 95-100% approximately 20S protein have been prepared for the first time and used for reconstitution with RNA. Such protein solutions yield full size TMV, but at a slower rate than if 4S protein is added. Thus the elongation reaction in TMV assembly, following nucleation with approximately 20S protein, is not exclusively dependent upon the presence of either 4S or approximately 20S protein aggregates. The initial, maximum, rate of reconstitution increases about threefold when the protein composition is changed from 5% to 30% 4S protein, at constant total protein concentration at pH 7.0, 20 degrees C in 0.10 M ionic strength K (H)PO4. The probable binding frame at the internal assembly nucleation site of TMV-RNA has been determined by measuring the association constants for the binding of various trinucleoside diphosphates to helical TMV protein rods. The -CAG-AAG-AAG-sequence at the nucleation site is capable of providing at least 10-14 kcal/mol of sites of binding free energy for the nucleation event in TMV self-assembly.     

A specific effect of calcium ion on the polymerization-depolymerization of tobacco mosaic virus protein.

Tobacco mosaic virus protein stored in the cold at low ionic strength between pH 5 and 5.5 is highly polymerized. When such protein is brought to room temperature and mixed with acetate buffer and additional electrolyte to give a final pH of 6.5 and ionic strength of 0.1, the protein is still in the polymerized state. When the temperature is dropped to about 5 °C, the protein depolymerizes rapidly, in the normal manner, if the added electrolyte is barium chloride, magnesium chloride, or potassium chloride. However, if it is 0.01 m calcium chloride, the depolymerization is slow, requiring about 12 h to reach completion. When the temperature of this depolymerized solution is raised, the protein polymerizes rapidly; when the temperature is dropped, the protein depolymerizes rapidly, just as in solutions free of calcium.Ion-binding studies show that calcium is bound to the protein during the initial step when it is brought to pH 6.5 and room temperature in the presence of calcium. The calcium is released during the slow depolymerization when the temperature is dropped and is not bound again during polymerization at pH 6.5, brought about by an increase in temperature. This means that polymerized protein at pH 5.5 has a structure capable of binding calcium ions, probably a helical structure like that of the protein in the virus. When pH is raised to pH 6.5 at room temperature, this structure remains long enough for calcium to be bound when present. These calcium ions stabilize the polymer, resulting in slow depolymerization when the temperature is lowered. When the temperature is raised at pH 6.5, a different, looser polymer structure is obtained, one not capable of binding calcium.     

Interactions of tubulin and microtubule-associated proteins. Conformation and stability of the oligomeric species from glycerol-cycled microtubule protein of bovine brain

1. The conformation of bovine microtubule protein prepared by cycles of assembly and disassembly in the presence of glycerol has been studied by near-u.v. circular dichroism (c.d.) over a range of protein concentrations. The effects on the conformational properties of ionic strength and of a pH range from 6 to 7.5 have been correlated with the known oligomeric composition of microtubule protein preparations, as determined by the sedimentation behaviour of this preparation [Bayley, Charlwood, Clark & Martin (1982) Eur. J. Biochem. 121, 579–585]. 2. The formation of 30S oligomeric ring species, either by decreasing ionic strength at pH6.5 or by changing pH in the presence of 0.1m-NaCl, correlates with a significant change in tubulin c.d. Formation of 18S oligomer by changing pH at ionic strength 0.2 produced no comparable effect. The c.d. of tubulin dimer itself is not affected by ionic strength and pH over the same range. 3. The results are interpreted as a small conformational adjustment between tubulin and specific microtubule-associated proteins on forming 30S oligomeric species, due to interaction with the high-molecular-weight-group proteins. The possible significance of this is discussed with respect to microtubule assembly in vitro. 4. By using this conformational parameter, together with equilibrium and kinetic light-scattering studies, the sensitivity of glycerol-cycled microtubule protein to dilution is shown to be strongly pH-dependent, the oligomers being much more stable at pH6.4 than at pH6.9. 5. Oligomeric complexes of tubulin with microtubule-associated proteins show marked stability under conditions similar to those for efficient microtubule assembly in vitro. Oligomeric material therefore must be incorporated directly during assembly in vitro from microtubule protein.     

Factors Influencing Conformation Changes in a 7S Protein of Soybean Globulins by Ultracentrifugal Investigations

Effects of pH, ionic strength, kind of salts and disulfide bond cleaving agent (2-mercaptoethanol) on conformation changes revealed on ultracentrifugal patterns of a 7S protein in soybean globulins were investigated. In the solution with lower pH than isoelectric point, this protein dissociated into two components in low ionic strength, but showed a 7S sedimentation pattern in higher ionic strength than 0.1. On the other hand, in the solution with higher pH than isoelectric point, this protoin showed aggregation to a 9S isomer in lower ionic strength than 0.1. Between ionic strength of 0.1 and 0.5, the mixture of 7S and 9S forms existed and in higher ionic strength than 0.5, the protein kept a 7S form stablely. These reactions were reversible and effect of 2-mercaptoethanol was scarcely observed but those of salts were observed.

The molecular weight of the 9S isomer was approximately 370,000 and the s_20,w value was 12.30S. Therefore, the 9S isomer was considered to be a dimer of the 7S protein.     

Purification and partial characterization of a collagenolytic enzyme from Pseudomonas aeruginosa.

A proteinase from Pseudomonas aeruginosa exhibiting collagenolytic activity was purified 1575-fold with a recovery of 24% by use of chemical and chromatographic technics. The enzyme preparation appeared to be homogeneous when subjected to chromatographic, electrophoretic and ultracentrifugational analyses. A standard state sedimentation coefficient of 2.10 S was calculated and further analyses indicated that the enzyme had a molecular weight of 17 500 and dimerizes under certain conditions to yield an apparent molecular weight of 34 000. In addition to insoluble collagen, the enzyme catalyzed the hydrolysis of congocoll, azocoll, soluble collagen and casein, but did not attack orcein-elastin, azoalbumin, p-toluene eulfonyl-L-arginine methyl ester, benzoyl-L-tyrosine ethyl ester, and the hexapeptide N-benzyloxycarbonyl-glycyl-L-prolyglycylglycyl-L-prolyl-L-alanine. Enzymatic activity against congocoll was 6-fold greater at pH 7.5 in Tris with HCl than in phosphate buffer at the same ionic strength. Cobalt, and to a lesser extent, Zn2+ appeared to activate the enzyme, especially in phosphate buffer. NcCN and p-chloromercuribenzoate did not appreciably inhibit enzyme activity, while (NH4)2 SO4, EDTA and cysteine displayed a significant inhibitory effect under certain conditions.     

Intracellular pH determination by a 31P-NMR technique. The second dissociation constant of phosphoric acid in a biological system

To evaluate the accuracy of pH determination by 31P-NMR, factors which influence the pK value of phosphate were appraised on the basis of the titration of 1 mM phosphate buffer solution. When the method is used for the determination of cytoplasmic pH, ionic strength is the major factor causing shifts of apparent pK (pK') value, and the magnitude of the shift can be predicted from the ionic strength calculated by means of the Debye-Hückel equation. Ions (Na+, K+, Mg2+, and Ca2+) and salivary protein affected the pK' value by 0.1 to 0.3 units in solution with a given ionic strength depending on the species of ion. The form of the titration curve varied with temperature. Based on these results, the value of 6.75 was obtained with the uncertainty of 0.12 for the intracellular pK' of frog muscle at 24 degrees C.     

Self-association of alpha-chymotrypsin at low ionic strength in the vicinity of its pH optimum.

The self-association of alpha-chymotrypsin and its di-isopropyl phosphoryl derivative in in I0.03 sodium phophate buffer, pH7,9, was investigated by velocity sedimentation, equilibrium sedimentation and difference gel chromatography. No differences between the native and chemically modified enzyme were observed in the ultracentrifuge studies, and only a marginal (0.6%) difference in weight-average elution volume was detected by difference gel chromatography of 5g/litre solutions on Sephadex G-75. From quantitative analyses of sedimentation velocity and sedimentation-equilibrium distributions obtained with iPr2P (di-isopropylphosphoryl)-chymotrypsin, the polymerizing system is postulated to involve an indefinite association of dimer (with an isodesmic association constant of 0.68 litre/g) that is formed by a discrete dimerization step with equilibrium constant 0.25 litre/g. In addition to providing the best fit of the experimental results, this model of chymotrypsin polymerization at low ionic strength is also consistent with an earlier observation that dimer formation is a symmetrical head-to-head phenomenon under conditions of higher ionic strength (I0.29, pH7.9) where association is restricted to a monomer-dimer equilibrium. It is proposed that the dimerization process is essentially unchanged by variation in ionic strength at pH7.9, and that higher polymers are formed by an entirely different mechanism involving largely electrostatic interactions between dimeric species.