X-RAY ANALYSIS OF PROTEIN DENATURATION
93 1
X-RAY ANALYSIS O F PROTEIK DENATERATION'*' MOXA SPIEGEL-ADOLF Department of Colloid Chemistry, D . J . McCarthy Foundation, Temple Cniversity School of Medicine, Philadelphia, Pennsylvania ASD
GEORGE C. HEXXY
Department of Physics, Temple University School of Medicine, Philadelphia, Pennsylvania Received January 28, 1941
The effects of protein denaturation vary according to the kind of protein and the nature of the denaturing agent (4, 6, 8, 11, 13). Reversibility of heat denaturation appears to be limited to serum albumin. No reversibility has been detected so far in other proteins submitted to heat denaturation nor in any protein submitt>edto ultraviolet light coagulation (5, 10). Furt'her differences in the changes brought about in proteins by heat and by light of short wave length become manifest in rotatory dispersion (9) and in optical absorption (17). Since especially the latter results seem to point to structural differences between heat-denatured and ultraviolet-light-denatured serum albumin, comparat.ive x-ray studies seemed warranted. This met,hod of investigation has the advantage t'hitt problems of solubility and dispersion do not interfcre with t'he result's. APPARATUS
The x-ray apparatus used in these studies has been described previously (15). The tube operates at 35 kilovolts peak with an average current of 15 milliamperes and is equipped with a copper target, aluminumfoil windows (0.013 mm.), and lead diaphragms (1 mm. in diameter and 47 mm. in effective length). The specimen is tightly packed into a hole drilled through a glass slide of 1.32 mm. thickness and is 88 mm. from the focal spot and 37.7 mm. from the film. An exposure time of 1 hr. was used in most cases. The moisture content of the cameras can be varied. The photometric evaluation of the x-ray diffract'ion patterns was done with the help of a photoelectric microdensitometer described by Spiegel-Adolf and Peckham (18). By using a special technique for an exact determinat.ion of the center of the diffraction patt'ern, the relative light transmissions 1 Presented March 14, 1940 a t the Meeting of the llmerican Society of Biological Chemists, held in Xew Orleans, Louisiana. * This work was aided by a grant to one of lis (M.Sp.-A) from the National Research Council Committee on Radiation.
932
MONA SPIEGEL-ADOLF AND GEORGE C. HENNY
( t ~ / t ~of) points in the diffraction rings are plotted against the distances from the center. .MATERIAL
Serum albumin was prepared from horse serum by ammonium sdfate fractionation and subsequent purification through repeated reprecipitation (eighteen times). Finally the protein was freed from salt by dialysis and electrodialysis. The 2 per cent solution had a pH = 4.57 and a conductance K = 9 X reciprocal ohms. The ash content of the protein was 0.7-0.85 per cent. The solutions mere dried in vacuo over phosphorus pentoxide to a final moisture content of 5.72-6.0 per cent. Microchemical analysis showed 7.70 per cent hydrogen, 47.86-48.18 per cent carbon, and 14.45-14.70 per cent nitrogen. The denaturations both by heat and by light were made in aqueous solutions of 1 per cent and 0.5 per cent, respectively. A 1 per cent serum albumin solution was kept for 15 min. in a boiling water bath. After a subsequent electrodialysis, the precipitation was practically complete. The precipitate was dried over phosphorus pentoxide in vacuo. The final moisture content was 5.85-6.13 per cent; the results of microchemical analysis gave 7.95-7.97 per cent hydrogen, 50.55-51.15 per cent carbon, and 14.68-14.73 per cent nitrogen. One 0.5 per cent serum albumin sample (sample A) was irradiated a t 25 cm. distance from the light source (Hanovia, D.C. 220 volts); another 1 per cent sample (sample B) was exposed (to the irradiation) a t a distance of 40 cm. In the latter case the temperature of the fluid never exceeded 29OC. After an irradiation of 2 hr., both samples still contained coagulable protein after the ultravio!et-light-denatured part had been removed through electrodialysis. The procedure was therefore repeated until sample A was practically free from coagulable protein, while sample B still contained heat-coagulable material. Sample A was evaporated to dryness at 100°C.; sample R was dried in vacuo over phosphorus pentoxide. Analysis gave the following values: moisture content, (A) 5.94-6.35 per cent, (B) 4.80-5.00 per cent; hydrogen, (A) 9.65-9.91 per cent, (B) 7.567.68 per cent; carbon, (4) 51.67-51.68 per cent, (B) 48.16-48.48 per cent; nitrogen, (A) 14.78-15.00 per cent, (B) 14.61-14.45 per cent. Sample A was distinctly yellow. For the x-ray diffraction exposures all samples were thoroughly ground in an agate mortar. In a first series of experiments, s-ray diffraction patterns were taken of the above mentioned samples of serum albumin. The diameters of the diffraction rings were converted into the corresponding planar spacings. Figure 1 gives a photometric graph of the diffraction patterns. The diffraction pattern of genuine serum albumin checks with values
933
X-RAY ANALYSIS OF PROTEIN DENATURATION
given in the literature (3): 4.51 and 10.19 A., instead of 4.5 and 10.2 A., respectively. These main spacings stay unchanged for the heat-denatured and the ultraviolet-light-denatured serum albumin. The only change in the x-ray diffraction picture of ultraviolet-light-denatured sample B is an apparent increase of diffracting material, as indicated by a uniformly lower light transmission of the film. This may be caused by a technical error in the packing of the substances or by chemical changes in the latter. Cleavage of the terminal groups, as observed even in cases of mild irradia-
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FIG. 1. Photometric transmission graphs of genuine serum albumin (unbroken line), 1ieat3-deiiatured (broken line), and ~iltraviolet-light-denatured(dotted line) serum albumin. t l / t o indicates relative transmission; min. indicates distance from renter of the x-ray diffraction pattern: A. indir:ltrn plnnar Ppacings (calculntrd by the Bragg equation) in Angstrom units.
tion of proteins (12), may cause a relativz increase of x-ray-diffracting nuclear groups. Changes in the water-binding capacity of prot,eins upon irradiation (7) may act) in the same direction. The decrease in the moist>urecontent of protein precipitated by ultraviolet light (4.80-5.00 per cent against 5.72-6.00 per cent,) seems to corroborate the latter assumption. The diffraction pattern of the heat-denatured serum albumin and of the ultraviolet-light-denatured sample A show sharpening of the backbone reflection (4.5 &) ; moreover, the heat-denatured sample and the dtraviolet-light-denatured sam le 4% show threp outer rings corresponding to spacings from 4.02-3.49 These la,tter findings are in good agree-
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934
MONA SPIEGEL-ADOLF AND GEORGE C. HENNY
ment with results reported by Astbury and Lomax (l), who describe in heat-denatured serum albumin a sharpening of the backbone reflection and “the appearance of at least one new reflection (3.6 A.).’, Contrary to previously reported findings in heated gelatin (3, 16), the changes observed in heat-denatured serum albumin are not reversible upon contact with water alone. Samples of the various protein powders were kept for 2 days in an atmosphere saturated with water vapor. The cameras during the exposure were changed into moist chambers. S o changes in the diffraction patterns were observed. From the above findings, differences may be inferred to exist between the products of heat denaturation and of ultraviolet light denaturation. Similar observations have been made before on gelatin exposed to heat and to x-rays (16). These differences may be only of a quantitative nature. Nevertheless, they seem t o point to the fact that there exist other types or degrees of protein denaturation besides the ones observed in heat denaturation. S e w evidence for the support of this theory has been recently furnished by Clark (2). Our results seem to obtain further confirmation from the changes observed in the x-ray diffraction pattern of ultraviolet-light-denatured sample A. This sample, which has undergone denaturation by both heat and ultraviolet light, shows an x-ray diffraction pattern practically identical with the one of the heat-denatured serum albumin. If heat and ultraviolet light denaturation would cause changes in the same direction, an intensification of the changes in the sample submitted to both kinds of denaturation should be expected. The absence of significant changes in the microchemical findings point in the same direction, although the more sensitive gold sol reaction indicates (12) differences in the cleavage products in heat and ultraviolet light denaturation of serum albumin. The conditions under which sample B was irradiated were such as to reduce the splitting of the protein to a minimum, but nevertheless caused irreversible changes in chemical, physicochemical, and immunological respects (14). Since such changes do not noticeably affect the x-ray diffraction pattern of serum albumin, this method does not seem promising for further studies on changes brought about in proteins by ultraviolet light. But since heat denaturation shows significant effects upon the x-ray diffraction patterns of serum albumin, our studies were continued in this direction. Upon the basis of the reversibility of heat denaturation in serum albumin, it mas assumed that the process underlying heat denaturation was a kind of ring formation and condensation. Astbury and Lomax (1) interpreted their x-ray findings in denaturation as a “marked coalescence of the main-chains by way of the backbone linkage.” This agreement seemed to warrant x-ray diffraction measurements on a sample of serum albumin in which heat changes had disappeared after a specific treatment with alkali.
935
X-RAY ANALYSIS OF PROTEIK DENATURATIOK
Former investigations had shown that only a t a certain ratio between heat-denatured serum albumin and sodium hydroxide a part of the protein goes into solution which after the removal of the electrolyte shows again the chemical, physicochemical, and immunological properties of the undenatured product. This same ratio was observed in the treatment of a part of the heat-denatured protein. To 0.28 g. of heat-denatured serum albumin n a s added 7 ml. of 0.01 A' potassium hydroxide. -%fter a pro-
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FIG. 2. Photometric transmission graphs of genuine serum albumin (broken line), of the soluble fraction (unbroken line), and the insoluble fraction (dotted line) of heat-denatured serum albumin after treatment with alkali. t l / t o indicates relative tpnsmission; mm. indicates distance from center of the s-ray diffraction indicates planar spacings (calculated by the Bragg equation) in Angstrom pattern; -4. units.
longed shaking, the electrolytes were removed by dialysis in running distilled nater. A soluble fraction n-as separated from an insoluble one and both were dried in a vacuum desiccator over phosphorus pentoxide. Xray exposures nere made in the usual way, but the exposure time was increased from 1 to 2 hr. A sample of genuine serum albumin was used as a control. Aistudy of the x-ray diffraction patterns of the alkali-treated insoluble fraction (fraction I) and of the alkali-treated soluble fraction (fraction 11)
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936
No91
SPIEGEL-ADOIIF
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of heat-denatured serum albumin s h o w tlic following changes: In t,he x-ray diffraction pattern of fraction I the sharpening of the backbone reflection has rcmainrd unchanged hut the three outer rings havc disappeared (figurc 2). In the x-ray diffract,ion patt.crn of fractiun I1 thc* sharpening of the backboue raflect,ionas well as t,he thrw outer rings are not noticeable (figrm 3 ) . Fract,ion I is truly denat,urcd aat,cr-iusoluble protein and yet loses upair iilliidi t,watnient tlic three outer diffraction rings. Fraction 11 is water soluble and, hesidcs losing the t i m e outer diffrart.ion rings, also s h o w a. loss of slitirpcning of t,lw haekboiie spacing ring. Therefore it ins that t.he loss OS blw thwc o u k r rings and t.he disappearance of the sliarponing of the backbonc reflrct,ion havc a different, meaning for the physicor:hcmical rhangw involwd in lieat. denatoration. The x-ray diffrai.tion pat,tcrri of fraction II looks very much like a diffractiori pattcrn of gcrriiinr~scrriin dhurnin. Sevcrt,helrss, slight (lifer-
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\;-my diliractiori imttwiw of ttic insoluhlc fraction (a) and tlie soluble fmclion (b) o f 1ir:it-den:itiired nlkali-tventrd ~ e m mnlbiisiin; ( e ) is t,he diffraction p a t t i i n of gcnuinc sci.tim :dhumiii.
eiiws still can ha iichced hetw-acn the diffraction patterns of fraction IT arid of i~ genuine serum albarnin. The former pattern seems on the wholp to bc IIW intensi: :ind show very Saint ring at a planar spacing of 2 A. r. I he inriming of thcw l.kX, G . .I. Chem. &rr. 1936, 866. (2) CI,ARK, d. H.: ,I. Gen. Physiol. 19, 199 (1935). (3) KATI, J. R. : Die Hontgenspektrngraphie nls l,.nfersuchirngsmethode, Handbuch der Riobgischen Arbeitsmethoden, rdited by E. Ibderhalden, Div. 11, part 3, Fasc. 6, p. 340. Urban and Schwarzenberg, Berlin-Wen (1934). (4) SPIEGEL-ADOLF, M.: Kolloid-Z. 38, 127 (1926). (5) SPJEGEL-.kDOLF, >I.: Biochern. z. 186, 181 (1927). (6) S P I E G E L ~ D OM. LF : Strahlentherapie , 29, 367 (1928). JI.: Ergeb. Physiol. 27, 832 (1928). (7) SPIEGEL-ADOLF, (8) SPIEGEL-ADOLF, M.: Biochem. z. 204, 1 (1929). (9) SPIEGEL-ADOLF, NI.: Bioohem. Z. 213, 475 (1929). (10) SPIEGEL-ADOLF, M Irch. Path. 12, 533 (1931). (11) SPIEGEL-ADOLF, M.: Biochem. Z. 262, 37 (1932); .I. Biol. Chem. 97, XLIV, (1932). (12) SPIEGEL-ADOLF, hl.: Biochem. J. 28, 1201 (1934). (13) SPIEGEL-ADOLF, M.:Biochem. Z. 170, 126 (1926). (14) SPJEGEL-.~DOLF, RI.: Med. Record 160, 430 (IC139). (15) SPIEGEL-ADOLF, hl.,A X D .HENSY,G . c.:J. Am. Chem. Soc. 61, 2178 (1939). (16) SPIEGEL-ADOLF, M., AND HENSY,G. c>.: Proceedings of the Thirty-third Annual Meeting of the American Society of Biological Chemists, .4pril26-29, 1939. (17) SPJEGEL-ADOLF, M.,A N D KRUMPEL, 0.:Biochem. Z. 208, 45 (1929). (18) sPIEGEL-.kDOLF, Jf., A N D PECKHAM, R . H.: Ind. Eng. Chern. 12, 182 (1940).
