March 20, 1957
EFFECTOF ?-RADIATION ON
IN C, 71.4; H, 6.6; N, Anal. Calcd. for C ~ ~ H Z S O(343): 4.0. Found: (2,713; H, 6.6; N,4.0.
.-
L-a-Phthalimido-p-phenylpropionaldehyde( L-VIIIa) L-IVa, 1.69 g., was converted into L-VIIIa by the same procedure used above for the corresponding DL-compound. The crude L-VIIIa was recrystallized from ligroin, b.p. 85-100’, to give 0.73 g. (43%) of L-VIIIa, m.p. 115-117”, [ c Y ] * ~ -157 D f 2” ( ~ 2 % in chloroform). Anal. Calcd. for C17Hl3OaN (282): C, 73.1; H , 4.7. Pound: C, 73.4. H , 5.1. DL-cr-Phthalmido-~-phenylpropionaldehydeEthylene Glycol Acetal (DL-IXa).-The procedure of Balenovif, et U L . , * ~ was employed to convert 4 g. of DL-VIIIa to DLIXa, 3.74 g. (81%), m.p. 108-109.5’ after recrystallization from hexane. Anal. Crtlcd. for C18H1701N(323): C, 70.6; H, 5.3; N, 4.3. Found: C, 70.4; H, 5.4; N, 4.3.
[CONTRIBUTION FROM
THE
AMINO
ACIDCONTENT O F
INSULIN
1305
DL-a-Benzamido-p-phenylpropionaldehydeEthylene Glycol Acetal (DL-IXd).-A solution of 1.08 g., 0.00333 mole, of DL-IXa in 40 ml. of methanol was heated for 3 hr. under refluxing conditions with 3.33 ml. of 1 M methanolic hydroxylamine and 3.33 ml. of 1 M methanolic sodium methoxide. The deep red solution was evaporated to dryness and the residue was extracted with hot ethyl acetate. The ethyl acetate extract was then allowed to react with 0.00333 mole of benzoyl chloride and 3.33 ml. of 1 M aqueous potassium carbonate, the ethyl acetate phase separated, dried over magnesium sulfate and evaporated to give 0.36 g. (36%) of DL-IXd, m.p. 118.5’. Anal. Calcd. for C18H190~N(297): C, 72.7; H, 6.4; N, 4.7. Found: C , 72.4; H,6.7; N , 4 . 7 . The authors wish to express their indebtedness to Professors C. G. Swain and J. C. Sheehan for discussions concerning the work reported herein. PASADENA 4, CALIFORNIA
QUARTERMASTER FOOD AND CONTAINER
INSTITUTE FOR THE
ARMEDFORCES]
Chemical Effects of Ionizing Radiation on Proteins.1,2 I. Effectof 7-Radiation on the Amino Acid Content of Insulin B Y MAURICEP. DRAKE,J. WALTER GIFFEE,DOROTHY A. JOHNSON
AND
VIRGIL L. KOENIG3
RECEIVED SEPTEMBER 14, 1956 This study was undertaken to determine the radiosensitivity of the constituent amino acids of a protein in order to provide information which might indicate the source of objectionable qualities of aroma and flavors that have been found to occur in the sterilization of food proteins by ionizing radiation. One per cent. insulin in basic (pH 8.5) and in acidic (pH 3.0) solutions was subjected to 0, 10, 20 and 40 million r.e.p. 7-radiation doses. Cystine, tyrosine, phenylalanine, proline and histidine are demonstrated to be very radio-sensitive. Leucine, valine, lysine and arginine are significantly destroyed a t the high irradiation dose level. The nitrogen-terminal amino acids of insulin, glycine and phenylalanine, are shown to be deaminated. Cysteic acid is identified in the hydrolysates of the irradiated insulin. An increase in molecular size of the irradiated insulin is reported.
Introduction A potential peacetime use of ionizing radiation is in the preservation of foods. Such use is predicated upon the solution of certain problems, one of which is the prevention or masking of disagreeable irradiation-produced odors and flavors. Although proteins have been identified as primary sources of these odors, no correlation between odors produced and amino acid content has been found.4 Irradiation of free amino acids has resulted principally in deamination,6 aldehyde production,6 H2S liberation from cy~teine/cystine,~ and hydroxylation and ring-splitting of aromatic amino acids and histidine.8 Only two previous investigations have attempted to determine whether amino acids making up the protein structure are destroyed by irradia(1) Paper No. 654 in series of papers approved for publication. The views or conclusions are those of the authors and are not to be construed as necessarily reflecting the views or endorsement of the Department of Defense. The mention of commercial products does not imply they are endorsed or recommended by the Department of Defense over similar products not mentioned. (2) Taken in part from a thesis submitted by M. P. Drake in partial ful6llment of the requirements for the M.Sc. degree, Dept. of Biochemistry, Northwestern University. Presented at Symposium on Radiation Sterilization Agr. & Food Chem. Div.. 130th Nat’l ACS Meeting, 1956. (3) Department of Biochemistry, School of Medicine, Northwestern University. (4) W. D. Bellamy and E. J. Lawton, Nrtclconics, 12, 141 54 (1954). (5) W. M. Dale, J. V. Davies and C . W. Gilbert, Biochcm. J., 46, 93 (1948). ( 6 ) G. Stein and J. Weiss, J . Chcm. Soc., 3256 (1949). (7) S. L. Whitchet, M. Rotheram and N, C . Todd, Narlconics. 11, [SI 30 (1953). (8) B. E Proctor and D. S. Bhatia, Biochcm. J,,I S , 1 (1953).
tion of the protein. I n one study,9 fish muscle irradiated by high-voltage electrons to a dose of 5.7 X IO6 r.e.p. (roentgen equivalent physical) showed no significant destruction of the “essential” amino acids. I n the other,l0 irradiation of aqueous Mserum albumin solutions with X-rays (44,600 and 66,900 r.e.p.) produced 30% destruction of glycine, alanine and glutamic acid, and 21, 18, 16 and 13% destruction of lysine, threonine, tyrosine and isoleucine, respectively. The chemical compounds responsible for the disagreeable irradiation-produced aromas and flavors are not known. A quantitative amino acid analysis of irradiated proteins of known character should indicate those amino acids in a protein which are most radio-sensitive. Further research effort could then be directed toward the characterization of the irradiation-breakdown products from those amino acids and their relation to the observed quality defects in some protein foods. Insulin was chosen as the first protein to be investigated because of its availability in a very pure form, and its characterization as to amino acid content.ll Since insulin does not contain cysteine, methionine or tryptophan, the necessity for hydrolysis in base and the interference of acid-hydrolysis breakdown products of tryptophan is eliminated, and cystine is left as the only sulfur-containing amino acid. (9) R. E.Proctor and D. S Bhatia, Food Tech., 4, 357 flR50). (10) E. S. G . Barron, J. Ambrose and P. Johnson, Radiolion Res., 2, 145 (1056).
(11) E. J. Harfenist, THISJOURNAL, 75, 5628 (1953).
139G
hI. P.DRAKE,J. \V.GIFFEE,D. A.
The dose necessary to sterilize foods has been indicated to be between 2 and 3 million r.e.p.I2 Higher levels of irradiation are necessary, however, to produce chemical changes of a magnitude large enough t o be measured by the analyses available. Four dose levels were used (0, 10, 20 and 40 million r.e.p.) so that curves of amino acid survival could be drawn. An additional Nz-flushed sample was irradiated a t 40 million r.e.p. for comparison of irradiation results with and without oxygen. Experimental Sample Preparation.-One per cent. itisu1iii1J solutions were used throughout this study. Aliquots (1-4ml.) were pipetted into 13 X 100 mm. Pyrex glass test-tubes, and the tubes sealed with an 0x5-gen-gas torch. Where NB-flushing was desired, the end of the test-tube was drawn out to decrease the opening size, and nitrogen bubbled into the solution through a capillary tube for 20 minutes before sealing. -Inalkaline-pyrogallol trap was used to remove residual oxygen from the tank nitrogen, and a water trap was used to prevent dehydration of the sample solution. For shipping and irradiation, test-tubes for each irradiation level were pIaced in a concentric ring near the center of a No. 2 tin can. The samples a c r e kept frozen a t all times until used, except for a 30-minute room temperature tempering period before irradiation, and during the irradiation itself. Thirty minutes had previously been determined as sufficient to thaw the samples in each can so that ice-protein concentration effects would not occur during the irradiation. Irradiation.-The irradiation was performed on contract by personnel of the Phillips Petroleum Company who opcrate the Nuclear Testing Reactor a t Arco, Idaho. The radiation source was y-rays from spent reactor-fuel rods. All irradiation doses were calculated to a point in the center of each can, and are expressed in r.e.p. The r.e.p. as used here refers to a 93 erg energy absorption per gram of material Phenyl Isothiocyanate N-Terminal Amino Acid Method.-011~-n11.sample aliquots (1 .T p moles insulin) mere used. Derivative preparation and procedure for chromatographic identification were those published by Landmann, Drake arid D i l l a l ~ a . ~ A~spectrophotometric method’j was used to determine that the derivatives mere pure. I n this method t lie :ilcohol-diluted 3-phenyl-2-thiohydantoin derivatives are read over tlie wave length interval of 256-275 mp, and the ratio optical density at 260 mplmax. optical density obtained. A derivative is considered pure if the ratio is less than 0.85. Carboxypeptidase C-Terminal Amino Acid Method .-One Iiuiidred-pl. sample aliquots (0.17 pmole insulin) were mixed in a 200-pl. pol>-ethylene beaker with 60 pl. (5% w-./ xv.) of c a r b o x y p e p t i d a ~ e . ~ The ~ solution was adjusted to PH 9 t)>-tlic addition of 10 pl. of 0.01 yor\;H40H, and the sample dr:twn into a capillary tube. After sealing, the samples were incubated a t 37” for 16 hours. The enzyme was inactivated b y iriimersion of the capillaries in boiling water for 5 minu t e s . T h e s:tinplcs were emptied into a 200-p1. beaker and tlrieti in ilnciio over PYO,. For chromatographic identificat i o i i , tile snniplcs were soluliilized bl- addition of 50 pl. of very dilute HCI solution. Five-pi. aliquots wcrc used in the ~iiialysisprocedure. Ultraviolet Absorption Spectra Analyses .-Two 200-pI. aliquots (0.34 pmole insulin) were taken from each sample, and each aliquot was diluted to 10 ml. with either 0.1 iV Na01-1 or 0.1 ,IrHCI. The samples were read over the wave length interval of 220-360 rnp on a Beckman spectrophotometcr. Correction formula for lion-aromatic ultraviolet absorptioii17: The slope ( k ) of the absorption occurring in the non(12) I
