1482 CHEMISTRY: D. RIDGEWAY PROC. N. A. S.

greatly indebted to Alvin Kwiram for much essential assistance with the magneticresonance instrumentation. We are also greatly indebted to George Pake for manyhelpful conversations.

* Sponsored by the National Science Foundation.t Alfred P. Sloan Fellow.t A. A. Noyes Fellow.§ Contribution No. 2878.1 Hausser, K. H., Z. Naturforsch, Ila, 20 (1956); Hausser, K. H., and H. Kainer, Z. Natur-

forsch, 9a, 183 (1954), Chem. Ber., 86, 1563 (1953).2 Elliott, N., and M. Wolfsberg, Phys. Rev., 91, 435 (1955).3 Duffy, W., Jr., J. Chem. Phys., 36, 490 (1962).4Edelstein, A. S., and M. Mandel, J. Chem. Phys., 35, 1130 (1961).5 McConnell, H. M., and R. Lynden-Bell, J. Chem. Phys., 36, 2393 (1961).6 Lynden-Bell, R. M., and H. M. McConnell, J. Chem. Phys., in press.7Turner, J. D., and A. C. Albrecht, Acta Cryst., to be published.8 The dimerization of WB and other free radicals has been discussed previously in another con-

nection by K. H. Hausser and J. N. Murrell, J. Chem. Phys., 27, 500 (1957).9 Sternlicht, H., and H. M. McConnell, J. Chem. Phys., 35, 1793 (1961).

10 Chesnut, D. B., and W. D. Phillips, J. Chem. Phys., 35, 1002 (1961).11 Chesnut, D. B., and P. Arthur, Jr., J. Chem. Phys., 36, 2969 (1962).12 McConnell, H. M., H. 0. Griffith, and D. Pooley, J. Cheni. Phys., 36, 2518 (1962).

DEPENDENCE OF THE ROTATORY DISPERSION CONSTANT X0 ONpH IN BOVINE SERUM ALBUMIN*,t

BY DoN RIDGEWAYt

GATES AND CRELLIN LABORATORIES OF CHEMISTRY AND NORMAN W. CHURCH LABORATORY OF

CHEMICAL BIOLOGY, CALIFORNIA INSTITUTE OF TECHNOLOGY

Communicated by Linus Pauling, Jul1y 2, 1962

In recent years, much attention has been directed toward the polarimetry ofprotein solutions. This has been the result of recognition that the protein mole-cule may occur in a stable helical conformation of specific geometry1 and the sug-gestion2-4 that the actual proportion of amino-acid residues in helical conformation,i.e. the helical content, in a particular protein may be simply related to parametersof the optical rotatory dispersion curve of that protein. Briefly,5 it has been found,upon examination of a large number of proteins and synthetic polypeptides, thatthe optical rotatory dispersion curves of these materials can be fitted very well bythe following relation :6

Fm'] = a (X2_X02 + b (X2_n) (1)

where [m'] = 3MR/(n2 + 2). [a]/100, n being the refractive index of the solution,Ml1R the mean molecular weight of the amino acid residues in the protein, and [a]the specific optical rotation, and where X is the wavelength of the light and a, b,and Xo are constants. It has been proposed7 that

a = laiR + faoH, b = f bo (2)

VOL. 48, 1962 CHEMISTRY: D. RIDGEWAY 1483

Here MaiR is the total intrinsic rotation of the protein excluding the contribution ofthe helix, and aoH and boH are constants characteristic of the a-helix and identical forall proteins. The factor f is the helical content of the protein.Although a and b have now been determined for several proteins, little has been

done toward evaluation of Xo and its behavior. Proper selection of the value ofX0 is, however, critical to the experimental interpretation. It has been shown8 ina statistical analysis of the problem of estimation of a, b, and Xo, first, that the valueof the parameter b is exceedingly sensitive to errors in that of Xo (whereas a isrelatively insensitive), and second, that for measurements above about 300 m/1a remarkably good fit to the data is obtained with equation (1) even with very largeerrors in the parameters. For these reasons, the present investigation was under-taken of the behavior of Xo in a system in which a protein possessing a reasonablehelical content is known to undergo a shape change in solution as a result of rela-tively simple changes in conditions.Bovine serum albumin was selected as a proper material for this study. Its

isoionic point is pH 5.05. As the pH is lowered, properties of the protein begin tochange at pH 4.3 and continue to change down to very low pH.9 Results frommany techniques have been interpreted'0' 11 to indicate that the acid change is anexpansion of the molecule from a relatively compact form. The polarimetricchanges are interpreted," if one takes Xo = 212 mu over the entire pH range, toshow a change in helical content from 48 per cent at the isoionic point to 30 percent at pH 2.3.Instrumentation.-The polarimeter employed in all measurements presented here was designed

by the writer and constructed in the Chemistry Instrument Shop of the California Institute ofTechnology.'3 Its principle is similar to that described by Gates.'4 The new polarimeter consistsessentially of a light source and monochromator, a polarizer, the sample cell, a Faraday cell, ananalyzer, and a photomultiplier tube detector. The Faraday cell is a solenoidal magnet, theaxis of which coincides with that of the optical system, with a column of water at its core. Thefunctioning of the instrument is based on the fact that matter becomes optically active whenplaced in a magnetic field, the magnitude and sense of the induced rotation being directly de-pendent on the magnetic field intensity. A measurement with the instrument consists in deter-mination, with fixed polarizer and analyzer, of that current which must be supplied to the Faradaycell exactly to cancel the sample rotation. The magnitude of the induced rotation, which is equalto the sum of that of the sample rotation and the instrument null, is then obtained from this cur-rent by reference to calibration data. Recognition of the condition of balance is based on amethod which utilizes the introduction of a 60-cps alternating current in the Faraday cell. Thiscurrent causes the plane of polarization of light incident on the analyzer to fluctuate symmetricallyabout the plane it would have in the absence of the ac field. For small fluctuations, it can be seenthat the intensity of light transmitted by the analyzer is proportional to (sin cot + a)2, where cois the circular frequency of the alternating current and 6 is the angle formed by the fluctuationaxis and the normal to the analyzer axis. But (sin cot + 6)2 = (1 + 262 + 46sinct - cos 2cot)/2.Thus the 60-cps component, 46sin cot, of the transmitted intensity and of the signal from thephotomultiplier tube is proportional to the angle 6 and vanishes when the fluctuation axis and thenormal to the analyzer axis coincide. The absence of a 60-cps component in the signal from thePM tube defines the condition of balance of the polarimeter. The null-detection and balancingsystems are arranged to detect any 60-cycle component and to rotate the fluctuation axis by varia-tion of the direct current in the Faraday cell until that component is reduced to the noise level.During operation, the instrument is maintained in the condition of balance electronically and thedirect balancing current recorded continuously.

There are two particular advantages to this type of polarimeter. The first is that one is con-cerned with the measurement of a current, a procedure capable of much greater precision than isassociated with the measurement of an angle directly. The second is that the instrument is a true

1484 CHEMISTRY: D. RIDGEWAY PROC. N. A. S.

null-detection device. The null condition has only to do with the presence or absence of a 60 cpscomponent and is not disturbed, for example, by either stray light or light scattered by the experi-mental solution.The sensitivity of the new polarimeter is +20 to 50 lsdeg, depending on wavelength. Its time

constant during most of the experiments described below was 0.5 see and could be decreased sub-stantially without difficulty. The workable wavelength range is 350-550 mIu. The wavelengthdrive speed is such that a dispersion curve over the range of the instrument is obtained withinabout 1 min. At this speed, the lag expected in a protein dispersion curve from time-constanteffects is well below the detectable level.The light source for the polarimeter is a magnetically stabilized Osram XB/501 high-pressure

dc xenon arc. The monochromator is a Bausch and Lomb 50 cm grating device blazed at 500 mnj.A 10 cm sample cell was employed in all experiments described below.Materials and Methods.-Crystalline mercury dimer of bovine serum albumin, mercaptalbumin

fraction, purchased from Pentex, Inc., was clarified by centrifugation, recrystallized, dissolved inwater, and passed over a mixed-bed ion exchange resin to prepare the salt-free, isoionic monomeraccording to the method of Dintzis.15 The final solution was concentrated to 2.5 weight % inprotein by evaporation at 20 and delivered in 1 ml amounts to a series of small screw-capped vials.The vials were frozen and stored at -20°. Material was not thawed until within a few hoursof use in an experiment, at which time vials were placed at 2° until completely thawed.

In all experiments reported here, the concentration of protein was 0.005-0.017c, the proteinconcentration being determined on intermediate dilution stages from absorbancy at 278 mMA. In1all cases, the final pH was attained by dilution of a small aliquot of protein into a large volume ofdilute HCl, so that the possibility of local high concentrations of acid was precluded. All dilutionswere made with class A Ostwald pipets. Solutions were used immediately upon dilution. Theprotein stock solution was held at 0° throughout the experiment; the acid solutions were at roomtemperature.

Serious variations were encountered until precautions were taken to minimize protein losses anddenaturation at the air and cell interfaces. It was found useful, first, to pipet in large dilutionsteps, so that the walls of the pipet could saturate with protein without significantly altering theprotein concentration. Second, mixing of the highly diluted samples prior to their being placed inthe sample cell was confined to teflon vessels. Third, the sample cell was pretreated with a subse-quently discarded aliquot of the actual sample for 5 min immediately before a dispersion curvewas taken. Finally, the cross-section diameter of the sample cell was considerably larger thanthat of the light beam so that any concentration changes which did occur at the surfaces during ameasurement would have to express themselves over some distance before they were seen by thepolarimeter. In spite of these precautions, concentration was a serious problem and the varia-tion from curve to curve (although not within a single dispersion curve) was large. It was never-theless considered desirable to work at these dilute concentrations in order to minimize any inter-molecular interactions that might affect the rotatory dispersion parameters.

Results and Discussion.-The maximum-likelihood estimate of Xo was determinedfor 153 dispersion curves in the pH range 2.0 to 5.4, with use of the alternate pro-cedure described by Ridgeway.8 Mean values of Xo at various values of pH areplotted against pH in Figure 1. It is seen that Xo remains nearly constant at about225 m~ufrom the isoionic point down to about pH 3.5 and that it then drops rapidlyto at least 180 m,4 at pH 2.0.The 153 values were fitted with a fourth-order polynomial by means of an ab-

breviated Doolittle procedure,'l0 and the analysis of variance was performed.The results are given in Table 1. As is seen from the table, the linear and quadraticterms are very significant, whereas the third- and fourth-order dependences are notsignificant. One must on this basis reject the hypothesis that Xo is constant andconclude that in serum albumin it shows both a linear and a quadratic dependence onpH under the present conditions.The observed dependence of Xo on pH has significance both to considerations of

VOL. 48, 1962 CHEMISTRY: D. RIDGEWAY 1485

250

240

S2300

220-

200 0290

ISO 0

I?0

igo0 0

La o LO pH 4.0 .0

FIG. 1.-Dependence of the rotatory dispersion constant Xe of bovine serum albumin on pH. Thecurve drawn is the cubic: y = -123 + 258x - 62.1x2 + 4.86x'.

TABLE 1ANALYSIS OF VARIANCE

Degrees of Sum of MeanSource of estimate freedom squares square F P*

Total 152 115,187Regression on x alone 1 5,700 5,700 8.45 0.005>Regression onx2after x 1 8,248 8,248 12.22 0.001>Regression onx3afterx2 1 2,018 2,018 3.03 0.10Deviation from cubic 149 99,221 665.91

* Probability that coefficient of respective component is zero.

the origin of the optical rotation of the a-helix and to the problem of estimation ofprotein helical content from polarimetric data.As to the origin of the optical rotation, it is clear from theory'7 18 that a, b, and

Xo are not themselves simple physical quantities. The optical rotation of a proteinconsists of contributions of the form of the first term on the right in equation (1)from at least two electronic transitions (each term containing, in place of Xo, thewavelength of the transition) as well as contributions arising from the mutualinteractions of these transitions which are not of this form. Equation (1) is form-ally the first two terms of the Taylor-series expansion of the theoretical expression.The parameters a, b, and Xo are weighted averages of analogous quantities appearingin this expression. The precise meanings of the parameters are defined as thosevalues which best permit approximation of the theoretical expression with equation(1).6 From the present state of theory, it appears very likely that the spectralproperties of the helix will predominate strongly over those of the randomly coiledpolypeptides in that averge which defines Xo. With these points in mind, it is pos-

1486 CHEMISTRY: D. RIDGEWA Y PROC. N. A. S.

sible for us to discuss qualitatively the significance of variations in the value ofxo.The optical properties of the polypeptide are largely dependent on the amide

chromophore. The isolated amide, the spectrum of which should be similar tothat of the randomly coiled polypeptide, possesses four electronic transitions inthe near ultraviolet,'9 an N V, at 185 m/i, an N V2 at 148 m1A, an n-7r* at220 mu, and a Rydberg transition at 166 mw. Orientation of amide groups intoa helical array produces, just as in a crystal, changes in these bands. Moffitt,20treating the a-helix in its response toward light as an exciton system, concluded thatthe correlative interactions in the helix would split the respective N -. V bands toproduce a component of axial polarization and a degenerate pair of shorter wave-length of radial polarization. Gratzer, Holzwarth, and Doty2l have confirmedexperimentally the predictions of Moffitt for the N -> V, absorption; they give 191and 206 m1A as the values for the radial and axial components, respectively. Theseauthors also observed a band at 222 m,4 in helical material, which they interpretedto be the n-7r* transition.

Moffitt considered almost the entire contribution to the optical rotation of thea-helix to depend on the N -* V transitions, neglecting any contribution fromthe n-wr* on the ground that its rotatory power would be very small. The 222miy band observed by Gratzer et al. has an oscillator strength somewhat larger thanthat assumed by Moffitt for the n-7r*. If it is the n-7r* band, then, as the authorspoint out, Moffitt's conclusion would have to be revised. Recent polarimetricobservations by Simmons et al.22 on a number of synthetic polypeptides and proteinsin the far ultraviolet, which showed an apparent negative Cotton effect with an in-flection point at 225 mi, provide very strong evidence for a large contribution tothe optical rotation from a band in this region. The present direct determinationof Xo at 225 miu in isoionic serum albumin both confirms the anomalous dispersiondescribed by Simmons et al. as the principal Cotton effect of the a-helix and indi-cates that the 225 m1.k band predominates in the optical rotation in the visible andultraviolet regions. These conclusions follow from the fact that Xo being an aver-age, would not otherwise be centered about any one of the bands.Simmons et al. observed that the Cotton effect at 225 my disappeared (which

is tantamount to a shift in X0 toward shorter wavelengths) when the proteins weretransferred to solutions containing high concentrations of urea. Such treatmeantis expected to disrupt the a-helix, and they concluded that the 225 m,4 band ispeculiar to the helix. A common property of no7r* bands suggests an alternativehypothesis for a shift in Xo toward shorter wavelengths either as the result of treat-ment with urea or in the present case of the acid expansion of serum albumin.Variations in Xo must be accounted for in terms of changes in either the relativerotatory powers or the positions of the spectral bands of the a-helix. Thus, forexample, the Xo-shift in serum albumin cannot be attributed to the blue shift in theN -- V bands produced by the helix-coil transition. Similarly, since the n-7r*band in simple amides is also found near 225 m1A, one would expect a contributionfrom this region to the average defining X0 (and a Cotton effect) in randomly coiledpolypeptides unless the rotatory power of the band is significantly decreased bythe helix-coil transition. no7r* bands are typically very sensitive to solvent effectsand are stabilized in water2 and by solutes such as urea. In interpreting the 222

VOL. 48, 1962 CHEMISTRY: D. RIDGEWAY 1487

mu band in polypeptides as n-7r*, Gratzer et al. pointed out that this transitionwould not show a solvent effect due to water in the oriented films with which theyworked because water was excluded from them. In an analogous way, hydrophobicbonding,24-26 essentially the condition that the protein be folded so as to minimizethe total nonpolar surface of the molecule in contact with water, produces regionsin the protein structure from which water is excluded. Such regions shoulddemonstrate the n-7r* band at 222 mjs even in aqueous solution.

It is proposed that the observed Xo-shift of serum albumin in acid and that ob-served by Simmons et al. in urea solution are produced principally by stabilizationof the nor* transition by water (or urea) in regions of the protein isolated from sol-vent in the isoionic state but exposed upon unfolding.

Tanford et al.26 have concluded as a result of their observations on f3-lactoglobulinthat changes in hydrophobic bonding affect the polarimetric properties of proteins.These authors have observed uncorrelated changes in the values of a and b in thisprotein to be produced by treatment either with acid or with organic solvents.They attribute such changes to effects on the intrinsic rotation 2aiR of the polarityof the environment of individual groups in hydrophobically bonded regions. Theydo not suggest a basis for such effects, although the example cited by them fromorganic chemistry involves resolution of conformational isomers as the result ofsolvent effects.Although it is reasonable that hydrophobic bonding should affect protein struc-

ture, no experimental method has yet been set forth that would provide some estima-tion of its extent in a particular protein. If the present interpretation should proveto be essentially correct, the behavior of Xo provides one with such a technique. Itwill therefore be of interest to see whether further study in other proteins indicatesa correlation of changes in Xo with changes in folding structure, as inferred from othermethods. In the particular case of proteins in which b is nearly zero, such as f3-lac-toglobulin, changes in a may provide another measure (see below).One test of the interpretation of the Xo-shift as a result of stabilization of the

no7r* band by water should be given by a comparison of Xo for a particular proteinin water and in solvents which have less effect on the n-7r* transition. It has al-ready been observed26' 27 that transfer from water to less polar solvents produceslarge changes in the rotatory dispersion curves of several proteins. These changeshave been interpreted as an increase in helical content arising from the greaterstability of internal (helix-forming) hydrogen bonds in the nonpolar solvents.However, the effect on the dispersion curve of a small change in Xo, if unrecognizedas such, would very likely be interpreted as a change in helical content. Sinceuntil very recently the error in determination of Xo was large enough to allow itto change significantly without detection, it would now be worthwhile to repeatthese experiments and examine Xo directly.

Concerning the general problem of determination of helical content from ro-tatory dispersion data, results have now been described in the literature for twoproteins which are incompatible with the model defined by equations (1) and (2).The one case is that of Tanford et al.25 with25 -lactoglobulin, in which large changes ina were produced which were accompanied by almost no change in b. The othercase, that of serum albumin, has already been pointed out, but interpreted incor-rectly, by Leonard and Foster.'2 These authors plotted dispersion data for serum

1488 CHEMISTRY: D. RIDGEWAY PROC. N. A. S.

albumin at several values of pH as a/X02b versus 1/Xo4b. Since one can eliminatef from a and b in equation (2) to yield

aoHa = b + X2a R,

equations (1) and (2) predict, as Leonard and Foster point out, that their plot willbe linear. Since the slope in such a plot is Xo2 2 aiR, it is expected, in light of thepresent results, that, although the data above pH 3.5 may be approximately linear,a transition will be found near pH 3.5 with the data from below this point approxi-mating, in the simplest case, a line of more shallow slope. This is preciselythe result obtained. (It is not clear why Leonard and Foster did not notice non-linearity in their Moffitt-Yang plots, although, as has been pointed out above, thenonlinear terms increase very slowly with increasing errors in Xo.)The applicability of equations (1) and (2) depends on specification of the values

of ao" and boH, presumably on the basis of measurements of proteins or syntheticpolypeptides which are completely helical, and on the assumption of a constant valueof the intrinsic rotation laiR over the entire range of experimental conditions.Serum albumin and other proteins which display a variation in Xo are examplesin which aH and boH cannot be determined. Tanford et al., whose data probablywould not permit recognition of significant changes in Xo as such (because of thesmall number of wavelengths), attributed their results to changes in the extent ofhydrophobic bonding, thus qualitatively the same interpretation as given here forthe serum-albumin system, and it will be of great interest to see whether Xo is con-stant in 13-lactoglobulin. If significant changes in the intrinsic rotation can occurwithout accompanying changes in Xo, then one will be able to detect them as pro-ducing deviations from linearity in the Leonard-Foster plot of a/Xo2b versus l/Xo4b.Since the contribution to LaiR of the individual amino-acid residues depends inpart on the spectral energies of the respective amides, it should reflect the blue shiftassociated with the helix-coil transition. In this case, laiR would always containan f dependence, as suggested by Leonard and Foster.Summary.-The rotatory dispersion constant Xo was determined for bovine serum

albumin at various pH in the region of acid expansion by means of a new, high-sensitivity polarimeter. Between pH 5.4 and 3.5, Xo was found to be approximatelyconstant with a value near 225 mM; below this pH, it decreased sharply to about 180m1A at pH 2.0. The observation confirms the anomalous dispersion in the regionof 225 mu to be the principal Cotton effect of the a-helix and indicates that the 225mu band, which is believed to be n-7r*, is the principal contributor to the opticalrotation of the a-helix. The Xo-shift upon expansion is interpreted to be the resultof stabilization of the no7r* transition by water in regions from wbich water is ex-cluded in the isoionic molecule. On this basis, study of the behavior of Xo is pro-posed as a method for estimation of changes in extent of hydrophobic bonding. Thesignificance of a variable Xo on estimation of helical content by means of parametersof the Moffitt-Yang equation is discussed.

The writer wishes to express his gratitude to Linus Pauling, who sponsored his fellowship at theCalifornia Institute of Technology, for his active interest and advice during the course of thepresent investigation. He wishes also to acknowledge J. H. Sturdivant and J. Vinograd for manyhelpful discussions.

VOL. 48, 1962 CHEMISTRY: D. RIDGEWAY 1489

* Presented at the 6th annual meeting of the Biophysical Society in Washington, D. C., Feb-ruary 15, 1962.

t Contribution No. 2867 of the Gates and Crellin Laboratories of Chemistry.$ U.S. Public Health Service Postdoctoral Fellow of the National Cancer Institute and Re-

search Fellow in Chemistry of the California Institute of Technology. Present address: Depart-ment of Biophysics and Biometry, Medical College of Virginia, Richmond, Virginia.

l Pauling, L., R. B. Corey, and H. R. Branson, these PROCEEDINGS, 37, 205 (1951).2 Cohen, C., and A. Szent-Gyorgyi, J. Am. Chem. Soc., 79, 248 (1957).3 Yang, J. T., and P. Doty, J. Amer. Chem. Soc., 79, 761 (1957).4 Schellman, J. A., Compt. rend. trav. lab. Carlsberg., Ser. chim., 30, 363 (1958).6 This general subject has been reviewed, e.g. by Ridgeway, D., in Advances in Biological and

Medical Physics (New York: Academic Press, in press, 1962), vol. 9.6 Moffitt, W., and J. T. Yang, these PROCEEDINGS, 42, 596 (1956).7 Doty, P., Rev. Mod. Phys., 31, 107 (1959).8 Ridgeway, D., presented at the 6th annual meeting of the Biophysical Society in Washington,

D. C., Feb. 15, 1962 (to be published).9 Tanford, C., J. G. Buzzel, D. G. Rands, and S. A. Swanson, J. Am. Chem. Soc., 77, 6421

(1955)."Yang, J. T., and J. F. Foster, J. Am. Chem. Soc., 76, 1588 (1954).1 Aoki, K., and J. F. Foster, J. Am. Chem. Soc., 79, 3385 (1957).12Leonard, W. J., and J. F. Foster, J. Biol. Chem., 236, 2662 (1961).13 The writer gratefully acknowledges the aid and cooperation of the Applied Physics Corpora-

tion, Monrovia, California, which donated the null-detection electronics to this project, and itspresident, Howard Cary, who first described the principle of this type of polarimeter to him.

4Gates, J. W., Chem. Ind., 77, 190 (1958).15 Dintzis, H., dissertation, Harvard University (1952).16 The writer wishes to express his gratitude to R. J. Monroe for his advice in the statistical

analyses.17 Moffitt, W., D. D. Fitts, and J. G. Kirkwood, these PROCEEDINGS, 43, 723 (1957)."8Tinoco, I., R. W. Woody, and K. Yamaoka, Tetrahed., 13, 134 (1961).1" Peterson, D. L., and W. T. Simpson, J. Am. Chem. Soc., 79, 2375 (1957).20 Moffitt, W., J. Chem. Phys., 25, 467 (1956).21 Gratzer, W. B., G. M. Holzwarth, and P. Doty, these PROCEEDINGS, 47, 1785 (1961).22 Simmons, N. S., C. Cohen, A. G. Szent-Gyorgyi, D. B. Wetlaufer, and E. R. Blout, J. Am.

Chem. Soc., 83, 4766 (1961).23Kasha, M., Disc. Faraday Soc., 9, 14 (1950).24 Tanford, C., in Symposium on Protein Structure, ed. A. Neuberger (London: Methuen and

Sons, 1958), p. 35.25 Kauzmann, W., in Advances in Protein Chemistry (New York: Academic Press, 1959), vol.

14, pp. 37ff.26Tanford, C., P. K. De, and V. G. Taggart, J. Am. Chem. Soc., 82, 6028 (1960).27 Doty, P., in Proceedings of the IVth International Congress of Biochemistry, Vienna (London:

Pergamon Press, 1959), vol. 8, p. 8.