INDUSTRIAL AND ENGINEERING CHEMISTRY Carter, Sage, and Lacey, Trans. Am. I n s t . M i n i n g JFet.
Engrs.,
142,170(1941).
Churchill, Collamore, and Kats, OiZ Gas J . , Aug. 6, 1942. Claude and Hess, Compt. rend., 124,626, 988, 996, 1000 (1897). Copson and Frolich, IND. EKQ.CHEM.,21, 1116 (1929). Dourson, Sage, and Lacey, Am. Inst. iMining Met. Engrs., Tech. P u b . 1490 (1942).
Furman (editor), “Scott’s Standard Methods of Chemical Analysis”, Vol. 11, New York, D. Van Nostrand Co., 1939. Guter, Newitt, and Ruhemann, Proc. R o g . SOC. (London), A176, No. 964, 140 (1940). Krauss, Azctogene Metallbearbeit., 28, 72 (1935). Kuenen, P h i l . Mag., 40, 173 (1897). Ibid., 44, 174 (1895).
Lamb and Roper, J . Am. Chem. SOC.,62, 806 Loomis and Walters, Ibid., 48,2051 (1926).
(1940).
Vol. 36, No. 7
(17) Maass and Wright, Ibid., 43, 1098 (1921). (18) McIntosh, J . P h y s . Chem., 11, 306 (1907). (19) Pickering, Ibid., 28, 97 (1924). (20) Porter, J . Am. Chem. Sac., 48,2055 (1926). (21) Quinn, So.D. thesis in Chem. E n g . , M.I.T., 1940. (22) Rimarski, Autogene Afetallbearbeit., 22, 134 (1929) (23) Rimarski and Konschak. Ibid.. 26. 129 (1933). (24) Rimarski and Konschak, Azetylen Wiss: I n d : , 33, 97 (1930) (25) Ibid., 35, 146 (1932). (26) Wolff, 2. angew. Chem., 11, 919 (1898). (27) Yee, Ph.D. thesis, Univ. Mich., 1936.
PRESENTED before the Division of Petroleum Chemistry a t the 107th hleetCleveland. Ohio. Abstract of thesis ing of the A Y E R I C A K CxEnrrcAL SOCIETY, submitted by J. L. McCurdy to Rackham School of Graduate Studies, University of Michigan, in fulfillment for Ph.D. degree.
PROTEINmALDEHYDE PLASTICS Reaction of Formaldehyde with Beaminized Casein E).
C . Carpenter and F . E . Lovelace‘
NEW YORK STATE EXPERIMENT STATION, GENEVA, N. Y.
T
HE first paper in this The combining ratios between formaldehyde and deaminthough it may not react its e r i e s ( 4 ) described ized casein are established over a concentration range up to self with aldehyde, greatly 6.85% formaldehyde. The general law, relating bound influences the constants remeasurements of the formaldehyde bound by acid formaldehyde to total formaldehyde, is shown to be the lated to the binding of the casein and rennet casein, and adsorption law, x KCn, Over the concentration range second mole of aldehyde. showedthat Over axviderange investigated. The value of n is the same for deaminized From the work of Dunn of aldehyde concentration, casein as for acid casein previously investigated. The and co-workers, we may exvalues for K are very different, only 45% as much aldehyde the aldehyde bound conclude that arginine binds being bound at any aldehyde concentration by deaminized pressed by the law X = KC”, casein as by acid casein. The aldehyde bound by acid One Of On the the constants having different at casein and deaminized casein agrees closely with that exwarnin’ group higher aldehyde concentravalues for the different capetted from the content of certain individual amino acids in the respective proteins. t i o n , o n e mole on t h e seins. Some 30% more aidehyde was bound by acid caguanidino group, H&Csein than could be accounted (=NH)NHR. The lattcr for by assuming reactions with a-amino groups, side-chain amino is indicated rather than the a-position by the fact that the Sakaguchi reaction (14) for the guanidino group is negative a f t e groups of the diamino acids, and amide groups. It was suggested aldehyde and arginine have reacted for some time. that the binding of aldehyde by hydroxyamino acids t o form With respect t o lysine, the €-amino group apparently reacts acetals should be considered a possibility, inasmuch as the interawith one mole of aldehyde before the a-amino group, and the tomic distances of the acetals involved were compatible with latter reacts eventually with the usual two moles of aldehyde. ideas of protein structure already obtained from x-ray studies. Histidine reacts with one mole of aldehyde at the a-amino Recent studies on aldehyde binding by amino acids by Dunn group and with a second mole a t higher aldehyde concentrations. and co-workers (8) and from this laboratory (6) have done much The great reactivity of the latter reaction leads one to suspect to clarify the general problem. We showed that the amide group that the second aldehyde may react with the imidazole ring of asparagine does not react with formaldehyde and that, in genrather than a t the a-amino as is usual. eral, the a-amino group reacts with one mole of aldehyde to give Proline reacts with only one mole of formaldehyde. The a fairly stable methylol derivative; the latter, in turn, reacts five-membered ring structure of proline and oxyproline makes it with a second mole of aldehyde to form a n unstable compound difficult to fit them into peptide chains according to modern ideas which gives up aldehyde readily and which is probably a n acetal of protein structure. The least difficulty is encountered by asof the type RNHCHgOCHsOH. This structure is indicated by suming proline t o occur a t the end of R peptide chain with the its properties and the fact that only in the presence of an alkyl imino group in peptide linkage. This rules out a reaction with group (such as in N-methylleucine) is the second hydrogen of the aldehyde amino group reactive with aldehyde. No reaction occurs with I n the first paper (4) we overlooked the possibility of aldehyde aldehyde when an acyl group is attached to nitrogen of the amino binding with the tyrosine side chain. However, Koebner (12) group ( 3 ) . The length and structure of the side chain, even prepared 2,6-dimethylol-4-methylphenolfrom p-cresol and forrn1 Present address. Curtico Bros. Co., Rochester, N. Y .
INDUSTRIAL AND ENGINEERING CHEMISTRY
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68 1
gen gas was employed t o blow out back-diffused air and thus reduce the formation of brown fumes to a minimum and their effect on protein caught in the foam. After deaminization for 24 hours at room temperature, no further deamination took place. The bulk of the supernatant liquid was removed with a siphon, and the deaminized protein separated in the centrifuge. The protein was washed repeatedly with water and separated in the centrifuge each time. Washing was continued with alcohol of increasing strength, until 98% concentration was reached, and completed with absolute benzene. The protein was dried at room temperature, spread out on a large sheet of plate glass, and dried in vacuo in a desiccator over PzOa to constant weight. The yield was 257 grams of dry, faintly yellow, deaminated product or about 90% of the original weight of casein employed. TABLE I. COMBINATION OF FORMALDEHYDE WITH DEAMINATED The total nitrogen of the original acid casein amounted to CASEINSAT EQUILIBRIUM (25' C.) 15.8%. This valge was lowered to 15.1% in deaminized sample Combined 1 (which was not completely deaminized) and t o 14.74 per cent FormaldeGrams Formin deaminized sample 2 (which would not react further with nialdehyde G.Combined Formaldehyde h @ o ~ ~ d ' Series per 1000 G. per G. Deaminated Casein 10-6) per q. trous acid). Sandstrom (15) reported 15.8% nitrogen in his origFound Calod. Deam. Caaein NO. Soln. inal casein and 15.09% in his deaminized casein. His analytical Deaminated Casein Sample 1 data showed unchanged lysine in the deaminized casein after 1 4.70 0.0111 0 01115a 37.3 0.01603 53.0 2 8.97 0 0159 hydrolysis; apparently his sample was about as completely de3 18.40 0 0251 0.02515 83.7 aminized as our sample l. 0.03694 123.0 4 35.10 0 0369
aldehyde, and it is probable that a similar 2,6-dimethylol derivative of tyrosine exists. The deamination reaction has been employed many times on proteins. Dunn and Lewis (6) and Sandstrom (16)deaminized casein and, after hydrolysis of the protein with acid, analyzed the hydrolyzed product by the nitrogen distribution method. They found that a-amino groups and the e-amino group of lysine were the principal groups affected, although some loss was noted for arginine and histidine. One of the Dunn and Lewis samples showed no further evolution of nitrogen by treatment with nitrous acid, and deamination was considered complete.
0.0550 0.05498 Deaminated Casein Sample 2 0.00797b 1 4.55 0.0080 0.01194 2 8.97 0,0120 3 17.46 0.0176 0.01776 4 35.10 0.0270 0.02886 5 68.50 0.0396 0.04004 From X = K C n where log K = -2.352 and n = 0.595. From X = KCn,'where log K = -2.490 and n = 0.595. 5
6
68.50
183.1 26.3 40.0 58.7 90.0 32.0
One of our deaminized caseins (sample 1) wa8 incompletely deaminized and showed more (sample 2), which evolved no sidered completely deaminized.
PROTEIN-FORMA LDEHYDE REACTION
The experimental method was described before (3); for each 5-gram sample of deaminized protein, 0.5 gram of calcium carbonate was added to maintain the p H a t 6.8. Formaldehyde solution (25 ml.) 9f the desired concentration was employed in each experiment. The experiments were set u p in 50-ml. centrifuge tubes corked with rubber stoppers so as t o be able t o separate the protein combined with aldehyde from the supernatant solution without another transfer. The supernatant liquid was removed after centrifuging and titrated with standardized sodium bisulfite solut e wet protein remaining in the tube titration so t h a t the s value subtracted from the aldehyde ori placed in the tube gave the aldehyde combined with the protein. Ten or twelve centrifuge tubes, identical with respect to contents, were set up sfor each aldehyde concentration series; a tube time intervals for centrifuging and ti-
and the 2,6-positions of tyrosine. Our results bear this out as closely as may be expected when one considers the variations in amino acid analyses reported by various workers. PREPARATION O F DEAMINIZED CASEIN
The same sample of crude acid casein employed befove was deaminated by essentially the method of Sandstrom ( I product was protected from light during deamination sequently, inasmuch as light causes the faintly yellow deaminated protein eventually to change color almost to a chocolate brown. The light yellow color is attributed t o the presende of nitro or nitroso derivatives formed during the deamination pr discussed previously. I n one of the preparations, 290 grams of dry acid casein were suspended in 5.8 liters of water and stirred mechanieally for several hours until swollen, and then 400 ml. of glacial acetic acid were added slowly. After further stirring, a freshly solution of 116 grams sodium nitrite in 250 ml. of water slowly beneath the surface of the casein solution, and stirring was continued overnight at room temperature. A stream of nitro-
Figure l. Formaldehyde Bound by Acid and Deaminized Caseins a t Various Aldehyde Concentrations
-
Acid Oasein, log K * 2.145 A Partly deaminized casein, log K = - 2,352 0 Completely deaminized casein, log K = -2.490 A _ - -
INDUSTRIAL A N D ENGINEERING CHEMISTRY
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Vol. 36, No. 7
correspond, respectively, to 49.1, 29.4, and 1.8 X lo-& mole of these hydroxyamino acids. Acid Casein ( 6 ) -Deaminized . )Casein 6 ( yo h' due Yo (Moles X 70 N due % (Moles X Since the main deamination % of t o amino amino pe? % of to amino amino 10-6) per total N acid acid gram casein total N acid acid gram protein reaction removes 16 grams of Arginine 7.42 1.172 3.66 21.0 7.09 1.069 3.32 19.1 NH, and replaces it with 17 6.01 0,950 3.52 22.7 3.89 0.586 2.16 14.0 Histidine grams of OH, the over-all change Lysine 9.09 1.438 7.49 51.3 0.67 0.101 0.53 3.6 Nitrogen (free in weight of the protein must NHI + lysine) .. ... 0.93 66.3 .. ... (none) 0.47 3.4 (none) have been small, and the amino acid analysis of the deaminated r Acid Casein ( 9 ) --Deaminized Casein (16)26.0 8.93 1.348 4.19 24.0 Arginine 9.20 1.455 4.53 protein not materially changed Histidine 6.26 0 99 3.66 23.6 6.41 0.817 3.02 19.5 except where a particular amino Lysine 8.49 1:3& 6.98 47.8 0.64 0.096 0.50 3.4 Nitrogen (free acid (lysine, etc.) has been reNHa + lysine) .. ... 0.96 68.6 .. ... .. .. moved. It seems allowable, 0 Nitrogen distribution analyses computed to basis of 15.8% N in acid casein and 15.09% N in deaminized therefore, for present purposes casein. to consider the content of certain amino acids to be unchanged by TABLE 111. ALDEHYDE BINDIXGO F A M I N O ACIDSIN the small change in weight due to deamination. Table-I11 is ACIDAND DEAMINIZED CASEINS drawn up on this basis, to show as clearly as possible the aldehyde Casein, (Moles X 10-6) per Gram of the various amino acids present in acid casein and binding Amino Acid Acid Deaminized deaminized casein. Dunn and Lewis (6) and Sandstrom (15) 26.0 Arginine 26.0 23.6 Histidine 23.6 record B 91 and 92% recovery, respectively, of arginine in the .., Lysine 47.8 deaminized protein and of 61.7 and 82% recovery, respectively, ... 41.6(X2) a-NHa ... 63.0(X2) Tyrosine of histidine. The former values are in good agreement and in49.1 Serine 49.1 29.4 29.4 Threonine dicate only a minor loss in arginine, but for histidine indicate a 1 . 8 8-Hydroxyglutamic 1.8 __ greater loss during deamination. The value recorded for histidine 129.9 Total 282.3 in deaminized casein probably should be revised downward to take this into account. Tyrosine loses a t least half and probably all of its ability to bind aldehyde because of the formation of nitro The tubes and contents were continuously shaken, end over end, and nitroso compounds. in a mechanical shaker immersed in a constant-temperature bath The binding of aldehyde a t an aldehyde concentration repreat 25' C. while reaching equilibrium. Because of the faint sented by log C = 1.800 is 281 moles X per gram for acid straw color of the centrifugate, the sulfite titrations were done casein, 175 for the incompletely deaminized casein, and 126.5 with a carefully standardized glass electrode against a saturated for the completely deaminized casein. The two latter figures calomel half-cell. correspond to 62 and 45%, respectively, of the aldehyde binding Table I gives the amounts of formaldehyde bound a t equilibof acid casein. The 282.3 total from Table 111 compares well rium by the two samples of deaminized casein a t the aldehyde with the experimental value of 281 X for acid casein, and concentrations reported, and the calculated amounts bound from 129.9 compares favorably with the experimental values 126.5 x the formula, 10-5 for completely deaminized casein. Correcting 129.9 for X = KCn losses in histidine during deamination would perhaps bring where X grams formaldehyde bound per gram protein even better agreement with experimental results. C = concentration of aldehyde K , n = constants for respective deaminated samples
--
TABLE 11. ANALYSES OF ACIDAND DEAMINIZED CASEINS"
.
.
LITERATURE CITED
Figure 1 shows graphically the binding of aldehyde by the samples, together with the aldehyde binding of the original acidcasein (dotted line). With all of the samples, deaminated or not, the value of n is 0.595 which shows that the rate of increase of aldehyde binding bears a constant relation t o increments in aldehyde concentration, The values of log K for acid casein, partly deaminated casein, and completely deaminated casein are, respectively, -2.145, -2.352, and -2.490. The significance of these constants is that each bears a relation to the number of places in the protein where binding of aldehyde may take place. The aldehyde bound a t any empirically chosen aldehyde concentration is 62 and 45%, respectively, for partly deaminized and completely deaminized casein, as compared with the original acid casein. BINDING OF ALDEHYDE
Table I1 gives analyses by the nitrogen distribution method of acid casein (6, 9) and deaminized casein (6, 15), as reported by different investigators. Analyses of acid casein for tyrosine have been reported as 5.70% (a), 4.50% (I), 7.49% (II), and 6.55Y0 (7). These values show little agreement but calculate to 31.5, 24.8, 41.2, and 36.1 x 10-6 mole of tyrosine per gram of acid casein. Recent analyses (13) of acid casein show 5.16% serine, 3.50% threonine, and 0.30% p-hydroxyglutamic acid. These values
(1) Abderhalden, E., 2. physiol. Chem., 44, 23 (1905); Hanke, M. T.,J . Biol. Chem., 66, 489 (1925). (2) (3) (4)
Bissegger, W., dissertation, Zurich, 1907. Carpenter, D. C., unpublished data. Carpenter, D. C., and Lovelace, F. E., IND.EKG. CHEM.,34,
(5)
Carpenter, D. C., and Lovelace, F. E., J . Am. Chem. SOC.,64,
(6)
Dunn, M. S., and Lewis, H. B., J. BioZ. Chem.,
759 (1942). 2899 (1942); 65, 1161 (1943). 49, 327-41
(1921). (7) (8)
Folin, O., and Ciocalteu, V.,Ibid., 73,627 (1927). Frieden, E. H., Dunn, M . S., and Coryell, C. D., J . P h y s . Chem.,
(9)
Hoffman, W., and Gortner, R. A., Colloid S y m p o s i u m Mono-
46, 215 (1942); 47, 10, 20, 85, 118 (1943).
graph, 2, 257 (1925). (10) Johnson, T. B., and Hill, -4.J., J . Am. Chem. SOC.,38, 1392 (1916). (11)
Jones, D. B., and Gersdorff, C. E. F., J . Bid. Chem., 104,
99
(1934).
(12) Koebner. M.. Anuew. Chem.. 46. 251 (1933). (13j Nicolet, 'B. N,, and Shinn,'L. '-4., J. BioZ. Chem., 140, Proc.
XCVIII
(1941).
(14) Sakaguchi, S., J . Biochem. (Japan), 5 , 25 (1925). (15) Sandstrom, W. N., J . Phys. Chem., 34, 1071-1101 (1930). PRESENTED before the Division of Biological Chemistry a t the 107th MeetSOCIETY, Cleveland, Ohio. Approved by ing of the AhfaRIcaN CHEMICAL the Director of the New York Experiment Station for publication as Journal Paper 588.
