4
PROTEIN-ALDEHYDE PLASTICS Combination of Formaldehyde with Acid Casein and with Rennet Casein D. C. CARPENTER AND F. E. LOVELACE ERHAPS less is known about the chemistry of proteinformaldehyde plastics than any of the other kinds of pphstic. The first of this type dates back to the formaldehyde-hardened casein material described by Spitteler and Krische (18). In 1900 the patent rights were sold to two Frenchmen, who produced the material under the trade name “Galalith”. The French and German interests merged in 1904 to form Internationale Galalith-Gesellschaft Hoff & Company. This firm became a large producer of caseinaldehyde plastic. In 1909 SchuItze secured a patent (16) for making a solid plastic from milk curd, and the product was marketed under the trade name “Erinoid”. The first successful casein plastic to be made in the United States is reported to have been developed by Christensen about 1919. Much of the casein-aldehyde plastic manufactured in this country reaches the market in the form of buttons or buckles. In any of the many variations for manufacturing caseinaldehyde plastic, the protein is eventually hardened by formaldehyde. It is this chemical reaction with which we are primarily concerned. The first work bearing on the combining ratio between formaldehyde and casein seems to have been that of Benedicenti (8) who reported that 0.0059 gram of formaldehyde combines with 1 gram of casein. He recognized that the reaction was a slow one, and his data are for a 16-day period of interaction with a 0.32 per cent formaldehyde solution. Ozawa (16) reported that 0.0057 to 0.025 gram of formaldehyde combines with 1 gram of casein; one free amino group supposedly binding one molecule of
New York State Experiment Station, Geneva, N. Y.
aldehyde. At higher concentrations a secondary type of binding also occurs. Gustavson (7) determined the formaldehyde bound by collagen a t pH 12. He concluded that the total aldehyde fixed by collagen under these conditions equals the sum of the lysine and arginine equivalents of the protein. He believed that aldehyde binding by the arginine residue does not contribute to the stabilization of the protein structure. Proteins are known to be composed of simple &-amino, diamino, and dicarboxyamino acids held together mainly by the peptide (CONH) linkage between the primary carboxyl and a-amino groups of different amino acids. Whether the structure of the protein molecule is of the chain type described by Astbury (1) or the cyclol structure deduced by Wrinch (% we! cannot I), decide at present. In casting about for simple model substances that would be expected to behave like the proteins toward the aldehydes, attention is called to the following: 1. The a-amino group of simple amino acids, whose reaction with formaldehyde was first investigated by S9rensen (17). The a-amino acids react with formaldehyde to give methylene derivatives: HCHO
+ HINCH?COOH glycine
4
+
CHpNCHpCOOH, Ha0 (1) methyleneglycine
2. The amino group in the side chain of the diamino acids, which would react with formaldehyde in the same way as the a-amino group above:
(CH34-NHa
The combining ratios between formaldehyde and acid casein and rennet casein are established over a concentration range up to 6.63 per cent formaldehyde. Acid and rennet casein are different from each other in the binding of formaldehyde. The general law applicable to both acid and rennet casein, relating bound formaldehyde to total formaldehydeemployed, is shown to be the adsorption law, X = KCn, in the concentration range investigated. The results indicate that some 30 per cent more aldehyde is bound than can be accounted for on the basis of combining with a-amino, side-chain amino, and amide groups. It is suggested that aldehyde combination with the hydroxy acids (serine, threonine, andb-hydroxyglutamic acid) of the protein may take place, and that acetal-like compounds will result which will or -O-CHyO-CH2-Oform -O-CH2-Obridges and thus unite the side chains of protein molecules. Trioxymethylene rings, formed from formaldehyde and enolized C 4 groups of the protein, may join protein plastic molecules together, further utilizing aldehyde. Models of the types of structure are given.
($%)rN=CHz
‘ I
HCHO
+ -N-b-C-
Llysine A8
1H methylenelysine ‘H 4 + HzO
+ -N-6-C-
(2)
3. The amide group in the side chain of dicarboxyamino acids, which would give methylene derivatives attached to the secondary carboxyl: CH&ONH*
HCHO
b-CA A b asparagine
+ -N-
--c
-N-
r=C -C-
LA 8
+ H20
(3)
methyleneasparagine
In Equations 2 and 3 lysine and asparagine are shown as their respective residues in peptide linkage as they occur in peptide chains. Methylene derivatives of the types mentioned have been prepared in the laboratory. Polymerization of methylene-amino compounds at the double bond may be expected just as with the more familiar vinyl plastics, although polymerization of the -CH2-NRunit would be expected to form derivatives of hexahydro-striazine, having alternate staggered carbon and nitrogen @tomsin a six-membered ring rather than as a linear polymer. This polymerization would take place for methylene-a-amino groups at the end of a peptide chain and unite three molecules 159
I
9
law
w
Vol. 34, No, 6
I N D U S T R I A L A N D ENGINEERING CHEMISTRY
760
0.06
2 0.04
9 0
FIGURE1. RATE OF BINDINGBETWEEN ACID CASEIKA N D FORMALDEHYDE
of protein together. Graymore (6) showed that CHFNalkyl or aryl compounds exist as trimers (CH-Nalkyl or aryl)l and preswnably have a cyclic structure, as will be discussed later. Polymerization of methylene-amino compounds at the double bond where the methylene group is attached to a side-chain amino is also possible, but steric hindrance would require that the amino acid carrying the side chain containing the methylene-amino group be located (for instance, for N omethylenelysine) no further from the end of the peptide chain than the length of two amino acid residues, This type of polymerization would link together three protein molecules at congruent corners. By determinations of the molecular weight, amino acid analyses, and serological studies, it has been shown that casein, as obtained by the precipitation from cow's milk by the addition of acid, is a mixture of three proteins having molecular weights of 96,000, 188,000 and 365,000 (29). I n our experience the bulk of the acid-precipitated material consists of the species of lowest molecular weight. The separation of crude casein by the method of electrophoresis has shown the existence of three species, so there is no reason t o believe that crude casein is other than a mixture of different proteins (11).
Preparation of Casein Samples Inasmuch as plastic is made from crude casein, the present investigation is concerned with experiments on a mixture of proteins which have undergone no process of separation from one another. Rennet casein was prepared from fresh skimmed milk (pH 6.8) by the action of commercial rennet; the curd was cut, well drained, thoroughly disintegrated, washed several times with water, and then squeezed fairly dry in a press, and the drying completed in trays in a current of warm air. The protein was then ground in a ball mill and sifted. The particle size passing through a 20-mesh screen and retained on a 40-mesh screen was used in the tests. The acid casein was recipitated from fresh skim milk by stirring in a fine stream ofnormal acetic acid solution until the H reached 4.6. The precipitated protein was filtered off, re&persed in water several times for washing, pressed, dried, ground, and sifted to size as above. Only that size of particles passing a 20-mesh and remaining on a 40-mesh screen was used in the tests. Moisture was determined on ap roximately 1-gram protein samples heated t o 61" C. in an Abgrhalden dryer over P,OS a t 1 mm. pressure. A sample was considered dry when the weight was constant over a 12-hour drying period. PROCEDURE. Five-gram samples of protein were weighed out and placed in a series of large test tubes; 25 cc. of formaldehyde solution of known concentration (which had reviously been exactly neutralized t o pH 7.0) were added to ea& sample, and rubber stoppers were adjusted so that a good sized air bubble was present t o assist in the subsequent stirring. After manipulating the tubes to ensure that no casein particles were stuck to the walls of the tube or otheiwise trapped the tubes were placed in a rotary shaker a t 25' C. and continuously shaken end over end. After various time intervals a tube was removed from the shaker,
the casein was manipulated to the bottom of the tube, and the liquid portion was removed to a flask for titration with standard sodium acid sulfite solution. The wet casein was weighed in the tube so that the titration could be corrected for the weight of the formaldehyde solution wetting the protein particles and held by ca illarity between the particles. %he sample of decanted unreacted formaldehyde solution was adjusted t o a faint pink color against phenolphthalein as indicator by the addition of sodium hydroxide solution, and then the formaldehyde was determined by the sodium acid sulfite method of Kleber (IO). Aldehydes are determined by this method with considerable precision (6). The analytical results on a single series of experiments are given in Table I for rennet casein. Figure 1 shows these data graphically. After a given time interval, the aldehyde bound by tthe casein has attained :t constant value; no further aldehyde will be bound except as conditions are changed, such as increasing the aldehyde concentration, etc.
U d 0.50
1.00
1.50
LOG C BOUND13Y ACIDAND RENXET FIGURE2. FORMALDEHYDE CASEINAT VARIOUS ALDEHYDE CONCENTRATIONS X For acid casein:
k = 2.145 n = 0.595
log
x
-
acid casein
=
KC"
--
For rennet casein: log IC 1,940
n
0.418
o = rennet casein
Table I1 gives data for several series embracing results for various aldehyde concentrations for both rennet casein and acid-precipitated casein. These results are shown graphically in Figure 2 and show beyond doubt that rennet and acid-precipitated caseins are very diaerent from each other. The general law for the casein-aldehyde reaction is expressed by the equation, X = KC" where X = weight of aldehyde bound per gram protein C = concentration of aldehyde, grams/liter K , n = constants which differ in magnitude for the two sorts of casein The single fact that the combining curves for rennet casein and acid casein cross each other shows that no matter how much alike the materials were in the original milk, they bear little resemblance to each other after rennet has acted on one of them. Most casein plastic manufacturers prefer rennet casein for fabricating their products. At low aldehyde concentrations rennet casein combines with more aldehyde than acid casein and, at aldehyde concentrations above 15 grams per liter, requires less aldehyde than acid casein. Some manufacturers prefer t o harden the material in baths containing 3 to 3.5 per cent formaldehyde while others prefer up to 10 per cent. Our data appear to show that quite different products would resulb by changing the aldehyde concentration if the reaction time was sufficient for
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
June, 1942
761 A
L
-
-
TABLE I. REACTION OF RENNET CASEINWITH FORNALDEHYDE (SERIES5 )
+
25 48 grams HCHO 0 59 ram Hz0 in cagein Sam le (Weight casein (dry) = 4.41 grams: weight solution used 26.07 grams total: formaldehyde concentration = 62.75.gram&l000 grams: 1 cc. NaHS8a = 0.03425 gram HC&O) Weights in Grams Grams of Formaldehyde Tube Tube.+ Tube.+ Soh. adsorbed Soh. oasein casein Time, empty Cc. NaHSOs Required NotTotal Combined Tube No. Days (dry) (dry) (wet) by casein titrated Uncor. Corrected combined combined per g. casein 1 25.48 47.80 18.47 44.45 2 5i:3 7160 3i:io 1.5227 o:iih 4+:3 59.9 0.0260 18.72 54.55 42.2 47.2 30.90 43.02 7.35 0.1636 3 1.4738 0.0371 18.67 41.68 7.4 0.2096 4 1.4277 59.3 46.9 51.9 29.85 0.0475 19.17 40.80 6.9 44.0 30.00 0.2399 5 1.3975 55.9 0.0544 49.0 19.27 6.8 50.6 29.77 40.40 0.2536 1.3838 57.4 0.0575 45.6 8 , 6 18.62 40.05 7.45 0.2655 1.3719 58.85 0.0602 51.4 28.65 10 7 46.8 18.92 39.75 7.15 0.2756 1.3618 60.25 0.0625 8 48.1 53.1 28.82 13 19.37 39.57 6.7 1.3554 53.3 29.40 0.2820 60.0 0.0639 9 48.3 16 1.3810 18.57 39.54 7.5 0.2864 60.0 0.0649 47.5 52.5 28.08 19 10 39.43 8.1 1.3507 57.0 0.0650 27.92 22 18.97 0.2866 43.9 48.9 11
...
....
....
i
TABLE11. COMBINATION OF FORMALDEHYDE WITH CASEINS AT EQUILIBRIUM (25’ C.) Series No.
Formaldehyde Concn. G./1000 G.’Soln.
Combined Formaldehyde, G./G. Casein Found Calcd. Rennet Casein
1 2 3 4 5
4.674 8.529 16.647 30.130 62.750
1 2 3 4 5
4.14 8.29 16.60 33.10 66.30
0.0217 0.0289 0.0367 0.0483 0.0649
Table 111 records the published analyses for certain amino
c $ ~ ~ $ ~ d ~ acids ~ ~ lobtained from acid casein. Results are included for (Moles X 1 0 d G . the diamino acids, free amino groups (which would include Casein
0.02138 0.02813 0.03720 0.04762 0.06477
96.5 72.3 122.3 161 .O 216.3
Acid-Precipitated Casein 0.0167 0.01664 0.0256 0.02521 0.03810 0.0374 0.05745 0.0574 0.0870 0.08686
55.4 85.3 124.7 191.3 290.0
terminal a-amino acids and lysine, inasmuch as this figure wm determined by decomposition with nitrous acid}, and amide groups (which would occur as CONHz groups in the side chains of dicarboxyamino acids). These would be the only data necessary to consider if the reaction between acid casein and formaldehyde is limited to the reactions given in Equations 1, 2, and 3. In columns 6, 7, and 8 of Table I11 the percentage composition of the various amino acids under discussion was calculated to a molecular basis. Each mole of nitrogencontaining compound would react with one mole of formaldehyde in hardening- the casein plastic. Although the discrepancies between the various reported analyses are wide, it appears from the more complete data in column 7 that 226 Of formaldehYde be the maxiI
to be established in each case. At all aldehyde
concentratioas the reaction proceeds rapidly for the hst two to four days and becomes about 90 per cent complete. Final equilibrium, however, is established slowly, probably due to the decreased rate of diffusion of aldehyde through the shell of hardened material surrounding the protein particle.
Discussion of Results
In the casein-formaldehyde reaction we are interested in what parts or groups in the protein molecule are undergoing reaction with the aldehyde, so that variations in the product may be intelligently followed and perhaps new products developed. The published amino acid analyses of acid casein show such wide variations between the results of different workers that it should have been obvious before our moleaular weight studies were made that different mixtures had been analyzed by these workers. Further, amino acid analyses are almost meaningless unless corrected for the destruction of the amino acid sought during hydrolysis. As examples, the recovery of tyrosine from acid hydrolysis is some 99 Der cent, while trvptophan - - - is comdetelv lost in acid hydrolysis and oily 81 per cent recovered in h ~ d r o l ~ sby is alkali. Cystine is completely lost in alkaline hydrolysis and 86 per cent recovered in acid hydrolysis.
TABLE111. AMINO ACIDS
IX
PARALLEL PEPTIDE CHAINS (ASTBURYMODEL) CONNECTED BY COHN (HYDROGEN-BOND) LINKAGES AND BY -0-CH2-OBRIDGEBETWEEN SIDECHAINS
FIGURE 3.
ACID CASEIN,ACCORDINGTO VARIOUSINVESTIGATORS
7,Amino Acid Histidine Arginine Lysine Free NHt (includes lysine) Amide Total
Vickery & White ($3) 1.83 3.85 6.25
.. .. ..
Hoffman & Gortner (8) 3.60 4.46 6.91
Osborne & Guest ( 2 4 ) 2.50 3.81 5.95
Van Slyke (BO) 2.53 3.59 8.38
0.96 1.52
1:32
..
....
..
..
Amino Acid (Moles X lO-S)/G. Casein Vickery & Hoffman & White (88) Gortner ( 8 ) Other investigators 11.8 23.2 22.1 25.6 42.8 47.3 58.1 (80)
.. ..
..
68.6 108.6 226.0
2:; [% E:: 82 194.2
INDUSTRIAL AND ENGINEERING CHEMISTRY
762
mum amount bound by 1 gram of acid casein. We found 290 x mole of formaldehyde bound by 1 gram of acid casein when the aldehyde concentration is only 6.63 per cent. The combining curve a t this point shows no evidence whatever of approaching a limit and in any case has exceeded the 226 X 10-6 figure by nearly 30 per cent. If we accept the analyses in Table I11 as substantially correct, it is clear from the present data that formaldehyde must combine in some way with protein other than at the places where a-amino, side-chain amino, and acid amide groups are located. The binding of more aldehyde than is accounted for by the reaction with nitrogen-containing groups may find an explanation in the combination of side-chain hydroxy groups of serine, threonine, and P-hydroxyglutamic acid residues with aldehyde in the formation of hemiacetals: H
H
I
H-L-OH I
H-C-0-CHzOH
+ HCHO
-N-h-C-
Lserine Lb
I
--c
-N-C-C-
I
I
(4)
A
H H hemiacetal
Acid casein has been reported recently to contain 5.16 per cent serine, 3.50 per cent threonine, and 0.30 per cent phydroxyglutamic acid (IS) which would account for an additional 49.1, 29.4, and 1.8 X 10+ mole, respectively, of aldehyde bound per gram of casein on the basis of hemiacetal formation'. The hemiacetal could then undergo further reaction with a second molecule of hydroxy acid t o give an acetal:
HzC\/OH
+ HOR'
-+
HzC\/OR'
+ HzO
Val. 34, No. 6
tioned is shown. The COHN linkage has a very low bond energy (2-6 K. cal. per mole) and is therefore easily broken while the C-0 linkage in the bridge has a bond energy of about 75 K. cal. per mole. Figure 4 shows two parallel peptide chains connected by COHN linkages in which the -O-CH2-O-CH2-Obridge connects the side chains of two amino acid residues in one of the peptide chains (left). An amino acid residue separates the two residues so connected. This type of linkage would be possible only on the side chains of alternate residues where the intermediate residue cont,ained no side chain to cause steric hindrancei. e., a glycine residue. The -O-CH2-Oand the -O-CHz-O-CH~-Obridges are equally applicable to Wrinch's cyclol structure. Holland (9) noted that collagen binds more formaldehyde than can be accounted for readily; he suggested that methylene cross linkages are formed between polypeptide chains of the protein, thus utilizing additional aldehyde. If the hydrogen-bond linkage is responsible for holding peptide chains together in Astbury's structure, as seems probable, it is pointed out that a -CH2group derived from formalde hyde cannot join together two arallell peptide chains for two reasons: The distance 4.65 i f is too great to be bridged group, and the bond angles are not right for by a -CHzsuch a union. Only when acetals are formed by the action of aldehyde on side chains, as described above, can the bond angles and interatomic distances be satisfied. Another equally probable type of linkage, holding together casein molecules in a plastic and involving a formaldehyde molecule as well as CON" linkages, will now be presented. Casein plastic is commercially prepared a t neutrality (pH 7.0)
(6)
OR
OR
hemiacetal
acetal
If the second hydroxy acid (R'OH) was contained in an adjacent peptide chain or in a second protein molecule, the two chains or molecules would then be connected by an -O-CHz-O-obridge. This type of linkage would give a distance of 4.67 A. between side chains so united. If the second hydroxy acid existed as a similar hemiacetal, the union between chains could be effected by the two hemiacetals reacting together: OH H,C
