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It has been suggested previously that water is a nearly indifferent solvent for amylose on the basis that the exponent a of the Mark-Houwink equation is very nearly 0.50 and the second virial coefficient A 2 is extremely small. It is shown that such solutions do not show the expected precipitation on cooling and the temperature coefficient of the intrinsic viscosity is small and negative. It is suggested that water is both an indifferent and a nearly athermal solvent for this polymer. Various lines of evidence suggest the polymer to exist as a very stiff coil in this and in other solvents. Variations in the intrinsic viscosity and radius of gyration in various solvents show the importance of skeletal effects in addition to the usual osmotic effects. Application of the Porod-Kratky wormlike coil model leads to unreasonably short persistence lengths if the contour length is based on a fully extended conformation but large and reasonable values of the order 40-70 A. if it is assumed that the basic conformation is the helical one observed in various amylose complexes in the crystalline state. As was previously known, the specific optical rotation decreases sharply in aqueous solutions above approximately pH 12. Accompanying this change in rotation there is first a decrease, then an increase in intrinsic viscosity. This later behavior is strikingly reminiscent of the known behavior of polyglutamic acid in the pH-dependent transition from helix to coil. It is concluded that the conformation is that of a stiff coil with essentially helical backbone contour in neutral solution, and the decrease in rotation and in intrinsic viscosity at high pH results from a partial breakdown of the helical structure with a concomitant increase in backbone flexibility. The increase in viscosity and radius of gyration in extreme alkaline solution is probably due to a polyelectrolyte expansion of the polymer coil. Some other miscellaneous observations which are in accord with this model are also pointed out.

Aqueous alkali has been assumed to be a good solvent for amylose, and has been used as a medium for physicochemical studies on this polymer. It is shown that there is a slow but substantial reduction in the turbidity of normal KOH solutions of amylose, even in the almost total absence of oxygen. More significantly there is a corresponding, but much more rapid, drop in turbidity in neutral solutions (pH 6.5–7.0). At pH 4.0 the turbidities are, in general, constant for up to at least 30 days. The results are interpreted on the basis of aggregates, which evidently must be of a very special type and are termed “native” aggregates. It is suggested that these may consist of amylose in essentially the helical configuration. The properties of these aggregates are in strong contrast to those of the retrograded aggregates. In the form of the first type of aggregate the amylose will not retrograde, at room temperature, without undergoing first a drop in turbidity suggesting that the transition from the “native” to the retrograded aggregate passes through the molecular state. The native aggregates are stabilized toward both disaggregation and retrogradation by pretreatment with iodine. Disaggregation is accompanied by a drop in relative viscosity. Some of the implications of these unexpected observations are pointed out.

In an attempt to clarify certain questions of the configuration of amylose in aqueous solution, carboxymethyl groups were introduced into corn amylose (degree of substitution ca. 0.8) and certain properties of the derivative (CMA) studied. The intrinsic viscosity exhibits a strong increase between pH 2 and 6 but remains constant at higher pH. A pH 6 and above, CMA exhibits typical polyelectrolyte behavior. CMA does not form a complex with iodine at pH 6 or above but does form a violet-colored complex at low pH. The absorption maximum of the CMA complex lies at a somewhat lower wavelength (550 mμ) than that of the complex formed by the parent amylose. The reduced viscosity and specific optical rotation of the CMA-iodine complex remains constant up to a concentration of 1.2 mg. iodine/100 mg. CMA, then both properties decrease with further increases of iodine content. All of the above results and hydrogen ion titration behavior are in agreement with the hypothesis that CMA exists as a coil of short helical segments in acid solution and that these helical segments break down on ionization of the carboxyl groups. The specific optical rotation of CMA increases between pH 2 and 6, remains constant between pH 6 and 12, and decreases above pH 12. This later result shows that the similar decrease in rotation seen in amylose cannot be attributed, at least entirely, to a helix-coil transition. There must be another effect on optical rotation, direct or indirect, of the ionization of hydroxyl groups.

Two statistical methods for the evaluation of the best values of the parameters of the Moffitt equation, λ0, a0, and b0, are proposed. The first is based on the maximization of the F ratio of the linear regression term to the residual term in the Moffitt-Yang form of the equation. The second is based on the minimization of the quadratic term in an extended form of this equation. Both methods are readily programmed for a digital computer. The usefulness of these equations has been demonstrated by analysis of a large number of dispersion curves obtained on polyglutamic acid through the region of the helix-coil transition and on bovine and human plasma albumins as function of pH. The results clearly show the inability of the simple Drude equation to fit dispersion data on native plasma albumin even over a relatively narrow range of wavelengths, and the remarkable ability of the Moffitt equation to fit data over a much broader range. It is shown that the best values of the dispersion parameters are not altered by extending the dispersion data from 300 mμ down to 240 mμ; however, precision is actually lost by this extension of the data. The best value of λ0 is shown to be constant within experimental error throughout almost the entire helix-coil transition in polyglutamic acid. The best value of λ0 is not affected by omission of the correction for solvent refractive index provided that the dispersion of refractive index is simple; however, the best values of a0 and b0 are appreciably affected by omission of this correction.

The molecular weight of flagellin, the subunit of flagella, from Proteus vulgaris was determined under various conditions by analytical ultracentrifugation employing the Archibald principle. In the absence of salt flagellin exists as a monomer (mol. wt. = ca. 20,000) below pH 3.8 and as a dimer at a pH above 4.5; the dimer is stable up to a pH of ca. 12. Trautman plots for flagellin in the transition region, and also time experiments indicate that the monomer-dimer equilibrium is attained fairly rapidly. An increase in ionic strength or a decrease in temperature increases the amount of dimer in the transition region. During the dissociation of the dimer a fairly large number of hydrogen ions ( q = 4–6) is taken up; this uptake can be most plausibly explained by the postulation that the ionization constants of carboxyl groups decrease as the dimer dissociates. The entropy of dissociation is strikingly similar to that of insulin and much smaller than expected on the basis of the simple increase in translational and rotational freedom. When the pH of flagellin solutions is lowered below pH 3.8, a change of optical rotation can be observed that is interpreted as an unfolding of the monomer accompanied by considerable loss of secondary and tertiary structure. This apparent unfolding is not prerequisite to the dissociation of the dimer which is essentially complete at pH 3.8 where the change in optical rotation is slight. In addition to monomer and dimer, under certain conditions flagellin also forms a rather stable seemingly spherical polymer. No aggregates of intermediate size have been observed in this study.

The results of a large number of streaming birefringence studies on dextran fractions, both native and degraded, produced by Leuconostoc mesenteroides are reported. The following solvents have been examined, all in admixture with varying proportions of glycerol (to enhance the solvent viscosity): water (with and without prior autoclaving), aqueous sodium hydroxide, formamide, and ethylenediamine. The results are surprisingly independent of the solvent used. Ethylenediamine–glycerol (equal parts by weight) was adopted for further investigation because of the high solvent viscosity. This solvent was found to yield a small birefringence with extinction angle near zero degrees, which, however, contributed no appreciable error except at polymer concentrations below 0.3%. Results were found to be substantially independent of polymer concentration and of temperature. Characteristically, the dependence of extinction angle on gradient was found to be much less than is to be expected for solutions of homogeneous, rigid molecules. It is concluded that this cannot be due to heterogeneity, at least in the case of certain well-fractionated preparation, and must be attributed to flexibility of the polymer molecule. It is shown that extrapolation of the apparent rotary diffusion constants to zero gradient can be carried out in most cases with some confidence. The resulting values, which should characterize the rotary diffusion properties of the undistorted polymers, are shown to be quite reasonable in terms of the known intrinsic viscosity and molecular weight of the fractions. The reduced birefringence of the various fractions is shown to be rather rigorously proportional to their intrinsic viscosity. Further, the extinction angles are found to approach a lower limit, at high gradient, which is related also to the intrinsic viscosity through the empirical equation Xlim = 24/[η]1/4.

The flow birefringence behavior of some amylopectins and their limit dextrins are reported. Though no quantitative interpretation is possible due to the great heterogeneity and their probable deformation in the flow gradient, several characteristics seem quite general. The extinction angle is relatively independent of the flow gradient. The gradient range over which observations could be made was limited at the lower end by lack of precision and at the higher end by precipitation which invariably occured. The birefringence curves characteristically showed a saturation behavior. Possible qualitative explanations for the observed behavior are discussed.

1. 1. Flow birefringence measurements have been made on solutions in which bovine fibrinogen (4 g./l.) and thrombin (1 unit/ml.) had been allowed to react for various times in the presence of 0.5 M hexamethylene glycol. 2. 2. The dependence of extinction angle on velocity gradient indicates the presence of elongated fibrinogen polymers of various lengths which increase with time of reaction, attaining values of the order of 4000 A. after 1 day. These polymers apparently dissociate into somewhat shorter particles when the protein concentration is reduced by a diluent which contains glycerol. 3. 3. The sign of the birefringence was in all cases positive. The magnitude of the reduced birefringence indicates that a substantial proportion of the fibrinogen is in the form of elongated polymers in these systems.

The behavior of bovine plasma albumin in moving boundary electrophoresis has been studied in great detail, especially by Aoki and Foster (l-6) and by Cann (7-10). In general terms, it has been found that this protein yields a single moving boundary at pH values above the isoelectric point (approximately pH 4.5) but two, and in some cases three, boundaries between pH 3.5 and 4.5. These results have been interpreted by Aoki and Foster (l-6) in terms of a cooperative alteration in the protein conformation, which takes place near pH 4. Some independent evidence for this “isomerization” has been obtained from solubility studies (11) and, more recently, from studies of optical rotation and rotatory dispersion (12, 13). It has also been suggested that the well known anomaly in the acid titration curve of this protein can be explained, in the main, as due to this phenomenon (6). At the time of initiation of the experiments reported here, no evidence existed for the isomerization of plasma albumins of any species other than bovine. It was shown earlier by Tanford, Swanson, and Shore (14) that the titration curves of human and bovine alhumins are essentially identical. This would imply that if indeed the titration anomaly in bovine plasma albumin is due to the isomerization reaction, the same reaction must take place in the human protein. However, Jirgensons (15) suggested that this may not be the case. We considered it of some importance, therefore, to examine the electrophoretic behavior of the human protein. Furthermore, since the plasma albumins are patently heterogeneous, in that some of the molecules contain free sulfhydryl groups and others do not, we were led to utilize the mercaptalbumin fraction of the protein by the thought that it might be more homogeneous. In the interim since this program was initiated, ample evidence has appeared that human albumin exhibits much the same type of isomerization behavior as bovine (12, 16-19). However, we know of no published electrophoretic studies that demonstrate the isomerization equilibrium in human mercaptalbumin nor, in fact, in the mercaptalbumin fraction of any albumin. A more important reason for presenting these results at this time is

Decreases in particle weight of amylose on aging in neutral and in alkaline aqueous solution are demonstrated by measurements of turbidity as a function of concentration. This further verifies that the previously reported turbidity drops are a consequence of disaggregation of an unusual type of aggregate. The same particle weights are obtained, in the case of aggregated samples, at pH 4 and in normal KOH. Anomalies arise in dilution series carried out in neutral solution and in solutions at pH 4 in the case of amyloses of low degree of aggregation, and a probable explanation is given. Samples can be partially stabilized in a given state of disaggregation by rendering the solution alkaline, and normal dilution series curves result. Disaggregation of the “native” aggregates continues along with retrogradation, and the retrograded aggregates are broken down readily by alkali. This leads to a method of disaggregation based on retrogradation followed by solution in 0.5 N KOH.

The effect of branching on the depolarization of light scattered from polymer solutions has been investigated employing various branched and unbranched polysaccharides. Two dextran fractions of M w 71,000 and 131 × 10 6 were studied in a strong solvent (aqueous KOH) and an indifferent solvent (water). The other polymers, studied in aqueous KOH only, included unfractionated corn amylopectin, the β-amylase limit dextrin of corn amylopectin, unfractionated corn amylose, and three subfractions of potato amylose (M w = 2.4 × 10 6 , 1.14 × 10 6 , and 0.27 × 10 6 ). Each polymer system was studied at several concentrations and all components of the scattered light, namely H u , V u , H u , V u , H u and V u , were measured in each case. In general the results are in agreement with predictions of Krishnan and of Doty. However, certain important deviations from predicted behavior are noted and discussed.

Studies of the rate of retrogradation (precipitation) of amylose from aqueous solution utilizing turbidity studies, are reported. A marked dependence of rate on the prior physical state of the amylose preparation is demonstrated. In particular it is concluded that retrogradation is retarded by increased perfection of the helical crystalline form. It is suggested that the rate of retrogradation is primarily governed by the rate of disruption of the intramolecularly hydrogen-bonded helical form of amylose. The process of retrogradation is thus akin to the prevalent picture of protein denaturation.

Hydrogen ion titration curves have been determined for two stoichiometric albumin-detergent complexes, AD11 and AD76, which are formed through interaction between bovine plasma albumin and the detergent sodium dodecyl benzenesulfonate. The amphoteric properties of the protein moiety in these two complexes have been compared with one another and with those of the uncombined protein. In this analysis, electrophoretic mobility data were used for making the necessary corrections for electrostatic interaction. Tyrosyl ionization curves were established for all three forms by measuring the change with pH of absorption at 295 mµ. The most significant differences found are in the tyrosyl ionization. For albumin itself, the apparent value of pKint is approximately 11.0, and it is concluded that some masked tyrosyl residues are exposed in a cooperative structural change accompanying the ionization. In the case of AD11, tyrosyl ionization is distinctly hypersharp and the apparent pKint is 11.3, implying that in this form the protein is stabilized against unmasking of the tyrosyl residues. In AD76 the apparent pKint is again near 11.0. The titration behavior of imidazole groups is virtually identical in the native protein and in the AD11 complex. In both cases it is necessary to assume two classes of imidazole groups in order to fit the titration curve. In AD76 the titration of these groups can be fitted adequately with a single pKint of 7.30. Surprisingly, very little difference is seen between the various forms in the titration of the e-amino groups. This fact permits the conclusion that electrostatic interaction between the detergent anions and cationic ammonium groups cannot contribute more than 700 cal to the free energy of binding, whereas the total binding free energy (on the unitary scale) must be of the order of 10,000 cal. The fact that the stoichiometry of the complexes is virtually unaltered over the pH range 6.9 to 11.6, over which range the total number of cationic sites in the protein falls from approximately 100 to 40, supports the conclusion that binding is not primarily electrostatic but is probably due to hydrophobic interactions. To a first approximation, all results suggest that the protein in AD11 is essentially in the native state and in fact is stabilized with respect to the structural alteration which must occur on conversion to AD76.

The airport must be integrated into community planning processes if its full potential for serving the community is to be realized. Towards this end, in 1967 the airlines instituted an airport facilities planning effort called the Airport Master Plan Report Program. These reports seek to identify airport improvements required for balanced airport capabilities in four broad categories: (1) Airport utilization— including air traffic control; (2) airport capabilities—including runway capacity; (3) surface transportation—on and off the airport; and (4) terminal facilities—passenger and cargo. The findings from the reports are presented to municipal planners, who in their turn, must develop a total planning concept which integrates airports into the life of each community and of the nation.

The Xenon lamp in a Cary 60 spectropolarimeter caused a photochemical reaction in proteins and polyamino acids which was traced to cleavage in the polypeptide chains. Viscosity studies on helical and coiled polyamino acids indicated that the cleavage occurred in both forms, but ORD changes were obtained only with helical and not with coiled and β conformations. Proteins of high helix content exhibited a significant change in ORD which was enhanced in the presence of denaturants. Quantum yields based on ORD change were greater in the n→Π ∗ than in the Π→Π ∗ transition region.

1. 1. Flow birefringence measurements have been made on inhibited clotting systems prepared by allowing bovine fibrinogen and thrombin to react in the presence of 0.5 M hexamethylene glycol and diluting the stock solution, which contains an intermediate polymer previously identified, to give various dilutions in 20 and 40% glycerol at constant glycol, salt, and buffer. 2. 2. The dependence of extinction angle on velocity gradient shows the presence of elongated particles of different lengths in the neighborhood of 4000 A., and the moderate dependence of extinction angle on concentration indicates that these particles do not disaggregate when solutions containing glycerol are diluted. 3. 3. The dependence of phase difference on concentration and velocity gradient also indicates that the particles do not change in length with dilution, at least down to a protein concentration of 1 g./l.