THE REACTION OF FORMALDEHYDE WITH PROTEINS* IV. PARTICIPATION OF INDOLE GROUPS. GRAMICIDIN BY HEINZ FRAENKEL-CONRAT, BEATRICE A. BRANDON, AND HAROLD S. OLCOTT (From the Western Regional Research Laboratory,j’ Albany, California) (Received for publication, December 11, 1946) In the study of the reaction of formaldehyde with proteins (2-4) it was noted that some of the aldehyde was bound in a manner not reversible by acid hydrolysis. Wadsworth and Pangborn (5) and Holden and Freeman (6) had previously found that histiclme and tryptophane differed from other amino acids in forming stable compounds with formaldehyde. Jacobs and Craig (7) and Neuberger (8) had prepared the formaldehyde derivatives of tryptophane and histidine, respectively, and found that new rings were formed by means of methylene bridges between the amino groups and reactive positions on the indole or imidazole rings! Recently Velluz (9) and Baudouy (10) suggested that the irreversible fixation of formaldehyde by proteins might involve their histidine and tryptophane residues. Similar ideas have been expressed by Nitschmann and Lauener (11) and Swain et al. (12). Gramicidin, t.he antibiotic isolated by Hotchkiss and Dubos (13) from tyrothricin, and shown to contain no polar groups other than indole and aliphatic hydroxyl groups (14), was found in this Laboratory to react with formaldehyde in neutral or alkaline solution to give a stable derivative of altered biological properties (methyl01 gramicidin) (15, 16). This, and other observations which will be discussed below, appeared to lend inde- pendent support to Baudouy’s claim concerning the irreversible fixation of formaldehyde by the tryptophane residues of proteins. However, as was pointed out by Swain et al. (12), the mechanism of the reaction could not be assumed to be the same as that for the free amino acid. The present publication is concerned with the conditions favorable for, and the mechanism of the combination of formaldehyde with, tryptophane residues in peptide linkage. The reaction with histidine residues is significantly different and will be discussed separately. The reaction of gramicidin with formaldehyde served as a model system and was studied in some detail, since gramicidin contains about 40 per cent * Presented in part before the American Society of Biological Chemists at Atlantic City, March, 1946 (1). t Bureau of Agricultural and Industrial Chemistry, Agricultural Research Ad- ministration, United States Department of Agriculture. 99 by guest on February 24, 2020 http://www.jbc.org/ Downloaded from
Transcript
THE REACTION OF FORMALDEHYDE WITH PROTEINS*
IV. PARTICIPATION OF INDOLE GROUPS. GRAMICIDIN
BY HEINZ FRAENKEL-CONRAT, BEATRICE A. BRANDON, AND HAROLD S. OLCOTT
(From the Western Regional Research Laboratory,j’ Albany, California)
(Received for publication, December 11, 1946)
In the study of the reaction of formaldehyde with proteins (2-4) it was noted that some of the aldehyde was bound in a manner not reversible by acid hydrolysis. Wadsworth and Pangborn (5) and Holden and Freeman (6) had previously found that histiclme and tryptophane differed from other amino acids in forming stable compounds with formaldehyde. Jacobs and Craig (7) and Neuberger (8) had prepared the formaldehyde derivatives of tryptophane and histidine, respectively, and found that new rings were formed by means of methylene bridges between the amino groups and reactive positions on the indole or imidazole rings! Recently Velluz (9) and Baudouy (10) suggested that the irreversible fixation of formaldehyde by proteins might involve their histidine and tryptophane residues. Similar ideas have been expressed by Nitschmann and Lauener (11) and Swain et al. (12).
Gramicidin, t.he antibiotic isolated by Hotchkiss and Dubos (13) from tyrothricin, and shown to contain no polar groups other than indole and aliphatic hydroxyl groups (14), was found in this Laboratory to react with formaldehyde in neutral or alkaline solution to give a stable derivative of altered biological properties (methyl01 gramicidin) (15, 16). This, and other observations which will be discussed below, appeared to lend inde- pendent support to Baudouy’s claim concerning the irreversible fixation of formaldehyde by the tryptophane residues of proteins. However, as was pointed out by Swain et al. (12), the mechanism of the reaction could not be assumed to be the same as that for the free amino acid. The present publication is concerned with the conditions favorable for, and the mechanism of the combination of formaldehyde with, tryptophane residues in peptide linkage. The reaction with histidine residues is significantly different and will be discussed separately.
The reaction of gramicidin with formaldehyde served as a model system and was studied in some detail, since gramicidin contains about 40 per cent
* Presented in part before the American Society of Biological Chemists at Atlantic City, March, 1946 (1).
t Bureau of Agricultural and Industrial Chemistry, Agricultural Research Ad- ministration, United States Department of Agriculture.
tryptophane, 5 times that present in the protein of highest known trypto- phane content. Simple indoles were used to substantiate the results with gramicidin. Finally, the conclusions were tested with proteins rich in tryptophane. The data indicate that 3-alkylindole residues react readily in alkaline solution with 1 equivalent of formaldehyde, which adds to the nitrogen or, less likely, the a-carbon atom to give a methyl01 group as
/ s /R
-0
Q
/ -c
I 11 \ N/C---H
I 11 \ N,C-CH20H
I Q
H CHtOH
(I) (II)
indicated in formulas I and II. The reaction is largely reversible in strong alkali but acid liberates only part of the formaldehyde and causes a break- down of the indole ring.
Methods
Formddehyde Anuly.s&-The amount of formaldehyde bound by a protein has usually been determined in the following manner (17, 18, 2, 3). The thoroughly washed protein derivative is subjected to a combined acid hydrolysis and distillation, formaldehyde being subsequently determined in the distillate by titrimetric or gravimetric procedures. These conditions, however, do not cause the release of formaldehyde from the methylene compound resulting from its interaction with free tryptophane, and only partial release of formaldehyde bound by the residues of thii amino acid in peptide linkage. Analyses for such “stably bound” formaldehyde have been based on determinations of the difference between the amounts added to, and recoverable from, reaction mixtures (12). However, it was found that even this technique could not be applied to tryptophane-containing products, since tryptophane derivatives react with formaldehyde under the conditions of combined acid hydrolysis and distillation (Table I) (11). The same difficulty was encountered when formaldehyde was determined by a chromotropic acid method (19) ; during t.he heating in strong sulfuric acid, indoles were found to bind formaldehyde.’ Thus it was not possible
1 Besides all indole derivatives, cysteine, dimercaptopropanol, tyrosine, and sev- eral proteins containing tyrosine and histidine bound formaldehyde under the con- ditions of chromotropic acid analysis (30 minutes in 14 N sulfuric acid at 100’) when 0.012 mg. of formaldehyde was used in the presence of 0.5 to 3.0 mg. of compound. On the other hand, histidine, acetylhistidine, histidine anhydride, and proteins low in tryptophane, tyrosine, and histidine (gelatin, isinglass, protamine) bound no appreciable amounts of formaldehyde under these conditions.
by either technique quantitatively to recover formaldehyde added to indoles after the sulfuric acid. In proteins rich in tryptophane, there appears to be no accurate method available for the determination of formaldehyde bound reversibly, or to determine by difference that bound irreversibly. Baudouy (10) and Nitschmann and Lauener (11) have also emphasized this difficulty. With Baudouy’s technique for irreversibly bound formaldehyde actually the sum of that ‘?rreversibly” bound plus that bound in so labile a manner as to be split off during the washing of the protein was determined.
TABLE I Irreversible Fixation of Formaldehyde by Indole Derivatives and Proteins under
Conditions of Analysis*
Substanck Formaldehyde bound per mole indole residue
* 5.7 mg. (0.19 mM) formaldehyde were added to 10 to 20 mg. of simple indole derivative or 100 mg. of protein in 50 ml. of 1 N sulfuric acid and distilled until fumes filled the distillation flask. Under the same conditions cysteine bound 0.45 equiva- lent of formaldehyde. Tyrosine, histidine, acylhistidines, and proteins free from, or very low in tryptophane (bovine serum albumin, globin, cattle hoof keratin, gelatin, insulin), bound no significant amounts.
t Distils with steam simultaneously with the formaldehyde. $ The insolubility and resistance to hydrolysis of gramicidin probably account
for its inability to bind formaldehyde under these conditions.
Special methods had therefore to be used in order to determine the extent of combination of formaldehyde with tryptophane in peptide linkage. The peculiar properties of gramicidin made it a most useful experimental material for this purpose. Its insolubility in water permitted its quantita- tive removal from reaction mixtures which could then be analyzed for residual formaldehyde, with or without distillation. The absence of amino, amide, and guanidyl, the usual polar groups that bind formaldehyde reversibly (2, 3), made it possible to attribute all formaldehyde bound by gramicidin after thorough washing to combination with its numerous tryptophane residues. The amount bound was ascertained (a) by differ-
ence between the amount added and that found in the combined super- natant and wash solutions, and (b) by elementary analysis of the derivative.
When simple indole derivat.ives and acyltryptophanes were used as model substances, the decrease in the formaldehyde content of reaction mixtures was determined directly, without distillation and without separation of the reaction product, by the addition of dimedon and buffer (20, 2, 3). Since the acylamino acid side chain binds no formaldehyde; the total amount bound by all model compounds used could be attributed to com- bination with the indole group. The data obtained by this technique were at times checked and confirmed by repeated precipitation and resolution of the indole derivatives with water and alcohol and ultimate analysis of the pooled supernatant solutions for residual formaldehyde. Character- ization of the modified reaction products or recovery of the unchanged indole derivatives supplied the final proof for the occurrence and the nature of the reaction.
Tryptophane Analysis-All 3-substituted indole derivatives, upon reaction with formaldehyde, lose their ability to give a blue color with Ehrlich’s reagent and give instead a purple color of lessened intensity if read with the usual red filter. This color test is similarly altered in form- aldehyde-treated proteins and hence supplies a quantitative estimate of the extent of reaction of their tryptophane residues. The analyses were performed according to Horn and Jones (22), either on unhydrolyzed proteins or on enzymatic digests (pancreatin). Alkaline hydrolysis could not be used, since it was found that strong alkali regenerated the chromo- genic activity. In addition, alkaline hydrolysis was found to cause some destruction of the tryptophane in chymotrypsinogen (Table IV) and lysozyme. Acid hydrolysis also causes destruction of tryptophane, particularly in the presence of formaldehyde.
Since gramicidm and methyl01 gramicidin are resistant to enzymatic digestion, these were analyzed either unhydrolyzed, in 50 per cent acetic acid solution, or After acid hydrolysis (16). Acid hydrolysis, even though performed in evacuated Thunberg tubes, led to formation of considerable amounts of humin in the case of methyl01 gramicidin (about 25 per cent of the nitrogen was rendered insoluble) but not with gramicidin itself, the tryptophane of which is resistant to acid hydrolysis with these precautions.
The Folin method for the determination of combined phenols and indoles was sometimes used as an additional technique. The buffer and reagent, prepared and diluted according to Herriott (23), were added simultaneously
2 As shown in many control experiments with several acylamino acids in the course of this study. Carpenter (21) and Neuberger (8) have demonstrated the inability of benzoylalanine and benzoylhistidine to bind formaldehyde.
to the appropriately diluted sample and the color was developed during 1 hour at 40’. By digestion of the protein with pancreatin (pH 8 for 3 days at 40”), the quantitative significance of the Folin values could be greatly increased, compared to those obtained on unhydrolyzed proteins. Direct analyses of intact proteins are known to yield values corresponding to only about 60 per cent of those expected; they have been found to be affected by the ease of denaturation of different derivatives (24,25). In contrast, the chromogenic activity of pancreatic digests of all proteins studied except lysozyme3 corresponded closely to the sum of their known tyrosine and tryptophane contents. This method of hydrolysis has an additional advantage in that it minimizes the danger of reversal of the modifying reaction.4
The formaldehyde derivatives of simple indoies and of chymotrypsinogen showed lowered chromogenic values with the Folin reagent. This observa- tion, however, did not facilitate the interpretation of the reaction mecha- nism, since the molecular requirements for a positive test are not known. Alkyl substitution of the indole ring did not greatly affect its chromogenic value with the Folin reagent. All indoles listed in Table IX gave from 50 to 70 per cent of the color of tryptophane, on a molar basis.
Other Anulyticul Techniques-Hydroxyl groups of gramicidin and methyl01 gramicidin were determined by quantitative acetylation (29, 14). Resorcinol and cetyl alcohol were 95 and 90 per cent acetylated under the conditions used. The solubility of gramicidin derivatives was estimated spectrophotometrically in a 25 per cent alcoholic medium (16). Nitrogen was determined by the modification of the Kjeldahl-Gunning-Arnold method recently described by White and Secor (30).
Matetials-Generous supplies of gramicidin were furnished by the Wallerstein Laboratories. Indole compounds, unless otherwise described, were commercial products. 2,3-Dimethylindole was synthesized by the method of Snyder and Smith (31). 1,2-Dimethylindole-3-acetic acid
3 Lysosyme had to be digested with pepsin after heat denaturation in acid solution (26) to release its complete chromogenic value (12.4 per cent, calculated as trypto- phane; see foot-note 6).
4 As an example, this technique has proved useful in establishing the number of phenol sulfate groups formed upon sulfation of proteins with concentrated sulfuric acid (27). An unhydrolyzed sample of insulin that had been sulfated for a short time at - 18’ appeared to contain 5.7 per cent tyrosine compared to the value of 8.9 per cent found with unhydrolyzed insulin. In enzymatic digests absolute values of 7.7 and 12.2 per cent were obtained, respectively. The latter value agrees with that obtained after acid hydrolysis. The former, when corrected for the amount of sulfate in- troduced, indicates that 30 per cent of the tyrosine had been sulfated. This product was found to have high hormonal activity (28). In contrast to the sulfate, O-acetyl- tyrosine was not stable under the conditions of enzyme digestion used.
was prepared from the methylphenylhydrazone of ethyl levulinate, as described by Degen (32), except that the condensation was carried out in glacial acetic acid and was catalyzed by boron trifluoride as described
TABLE II Rate of Reaction of Gramicidin and Indole Derivatives with Formaldehyde at
* The reaction conditions were as follows: 1 mM of the indole derivative or 200 to 400 mg. of gramicidin were treated with 5 to 7 mM formaldehyde in a final total volume of 10 ml. Sodium hydroxide, phosphate buffer, pH 8.0, or acetic acid was added to final concentrations of 0.03 M, 0.14 M, or 0.15 M respectively. With the exceptions noted in foot-note 5 the medium contained 4 to 6 ml. of alcohol. At the end of the specified time period, 1 ml. of the reaction mixture was diluted first with 3 ml. of alcohol and then with water to 10 ml. 3 ml. of the diluted solution were added to a mixture of 50 ml. of dimedon solution and 75 ml. of acetate buffer (20). After 24 hours, the precipitate was filtered, dried, and weighed. If contamination of the precipitate with the indole reaction product was suspected, nitrogen analyses were run. The results were uniformly negative. With gramicidin, the reaction product was precipitated by the addition of aqueous 0.1 M sodium chloride. After being centrifuged and carefully washed, the product was dissolved in alcohol and again precipitated and washed, The combined supernatant solutions and washings were analyzed for formaldehyde by the dimedon method.
t Measured after dilution with 2 to 3 volumes of water. $ That the reaction was practically complete in 2.5 hours was also indicated by the
fact that this preparation contained only 20 per cent of the hemolytic activity of gramicidin in vitro (15, 16).
5 In aqueous media. Indoleacetic acid was insoluble at pH 3.5 but gradually went into solution during the course of the reaction.
for other indole compounds by Snyder and Smith (31). The properties agreed with those in the literature. Chymotrypsinogen was a commercial preparation that had been recrystallized eight times by E. F. Jansen.
* In most cases, 4 to 20 per cent formaldehyde solution and 1 N acid or 0.1 N alkali were added to 400 mg. of gramicidin dissolved in alcohol, in order to give the desired end-concentrations, and the mixture was diluted to 10 ml. The alcohol concentra- tion was 40 to 60 per cent. Reaction time, 2 days at 53” or 70”, 7 days at room tem- perature.
t Antibacterial activities were as follows: the products prepared at 70” and 53” in acetic acid <lO and 73 per cent, respectively; that prepared at neutrality, 93 per cent; and the products of complete reaction in alkali, 81 to 96 per cent of the activity of gramicidin. The hemolytic activities in vitro were 10 to 15 per cent for the latter preparations and 50 to 100 per cent for those prepared at 53” at neutrality and in acetic acid. The methods of assay were those described elsewhere (11).
$ Analyzed without hydrolysis in 50 per cent acetic acid solution; approximate values. An atypical purple color was obtained in all samples designated as contain- ing <8 per cent.
$ By quantitative acetylation (19). 11 In 25 per cent aqueous alcohol containing 0.125 N sodium chloride (12). 7 Gramicidin exposed to strong alkali showed slightly lowered N (14.2 per cent)
and unchanged tryptophane contents. Treatment with acetic acid at 70” gave a pro- duct containing 14.2 per cent N and 34 per cent tryptophane.
DISCUSSION
Extent of Reaction-The data in Table II show that gramicidin in alkaline solution reacts rapidly with formaldehyde, binding almost 1 equivalent
for each tryptophane residue. The reaction proceeds rapidly at room temperature and at low formaldehyde concentrations in the presence of 0.02 N or higher concentrations of alkali (Tables II and III).6 In neutral solution little formaldehyde appears to be bound. In acid solution the reaction proceeds only at high formaldehyde concentrations and temper- atures.
In contrast to gramicidin, acyltryptophanes and simple 3-substituted indoles react at room temperature in acid and neutral as well as in alkaline solution, although at somewhat slower rates (Table II). Like the trypto- phane of gramicidm, these model substances bind almost 1 equivalent of formaldehyde.
Tryptophane analyses of formaldehyde-treated chymotrypsinogen, a protein particularly rich in this amino acid6 (33), demonstrated that the indole groups of a typical protein, like those of gramicidin, react rapidly and even at low formaldehyde concentration in alkaline solution, but not appreciably below pH 8 to 9 (Table IV). Similar results were obtained with tobacco mosaic vinis (Table V) and lysozyme, although the insolu- bility of the alkaline formaldehyde reaction products of these proteins prevented as complete a study as was possible with chymotrypsinogen.7
6 Previously, elevated temperatures (53”) had been used for the gramicidin-form- aldehyde reaction (15). The instability of formaldehyde in warm alkaline solution and the possible effect of indoles on its stability in such solutions rendered quanti- tative work at 53” difficult to control. Main emphasis has therefore been placed in this publication on the more reliable results obtained at room temperature. How- ever, the properties of methyl01 gramicidin are the same, whether prepared at room temperature or at 53” (in alkaline solution).
6 Of all proteins studied, lysozyme alone had a higher tryptophane content, that, is, 8.0 per cent, corrected for moisture, when analyzed (22) either unhydrolyzed or after enzymatic digestion. After alkaline hydrolysis or acid hydrolysis in evacuated Thunberg tubes, about 4.6 per cent tryptophane was found, compared to 3.8 and 3.3 per cent for chymotrypsinogen under the same conditions. Gramicidin, after acid hydrolysis in Thunberg tubes, appeared to contain 38.1 per cent tryptophane.
7 The finding of Ross and Stanley (35) that formaldehyde treatment at pH 7 re- duced the Folin color of unhydrolyzed tobacco mosaic virus was confirmed. How- ever, tryptophane analyses (22), both on the intact protein and after enzymatic digestion, indicated that there was no appreciable difference between this derivative and the original protein (2.5 to 2.7 per cent), while the formaldehyde derivate pre- pared at pH 11 contained less than 1 per cent unchanged tryptophane. It appears that the Folin color of the intact protein, which corresponds to only 53 per cent of the potentially reactive groups, is decreased non-specifically after formaldehyde treat- ment. Miller (25) found this to be the case for certain acyl derivatives of the virus unless they were previously denatured. Analyses with the Folin reagent after enzy- matic digestion confirmed the tryptophane analyses in showing that there was no decrease in chrofnogenic activity after formaldehyde treatment at pH 7, but a marked decrease after treatment at pH 11. Ross and Stanley’s results are thus to be in- terpreted as reflecting a decreased availability of the tryptophane and tyrosine residues to the Folin reagent after formaldehyde treatment at pH 7.0 rather than
Reaction of Tryptophane Residues of Chymotrypsinogen with Formaldehyde
per cent
I 1 1 1 1 1 1 5 5
10 10 10 10 10 10
None Control
Reaction conditions’
Final pH
11.5 10.1 9.1 8.8 7.6 7.5 6.1
11.1 7.4
10.5 8.2 7.3 6.0 3.8 2.8
11.7
B&3
None Phosphate None Borate11 None Phosphate None
<‘
Phosphate None
I‘
Phosphate None
“ “ 6‘
I Unhydrolyzed
per cent <O.S§ <0.7§
4.3 4.9 5.8
Insoluble ‘I
<0.6§ Insoluble <l.O§
3.9 5.0
Insoluble 6.1 5.1 6.0 5.8
-
Analytical conditions
Tryptophane -
I -
-
Enzymatic Alkaline dig&t hydrolysate
per cent
<2.3$ <3.1§
3.8 4.2 5.0 4.8 5.4
<2.6$ 4.2
<2.0§ 3.3 3.9 3.4
5.3 5.87
J%r cent
3.3
3.4
3.4
3.8 3.8
Phenol + indolet
Enzymatic dig&t
per cent 4.7
4.6
4.7
9.0 10.1
* 50 mg. of chymotrypsinogen were allowed to react in a total of 3 to 4 ml. When buffers were used, these were present in approximately 0.1 M concentration. Other- wise the pH was adjusted with I N sodium hydroxide or 1 N hydrochloric acid. After reaction for 1 day at room temperature, the protein was isolated by dialysis and dried by lyophilization. The yields averaged 90 per cent. Rate studies indicated that, at pH 11 to 11.5, the reaction had already proceeded to completion in 1 hour with formaldehyde concentrations of 0.3 or 1.2 per cent.
t Prepared as follows: To 5 to 10 mg. of the protein or protein derivative were added 1 ml. of HzO, 0.1 ml. of 3.4 M phosphate buffer (pH 7.6), and 0.5 ml. of a centri- fuged aqueous extract (10 ml.) of 10 mg. of commercial pancreatin. The mixture was held at 40” for 3 days, and then diluted to 10 ml. If solids were still present, they were removed by centrifugation, washed, dried, and weighed. Aliquots of the soluble fraction were subjected to calorimetric, and generally also Kjeldahl, analyses.
$ Calculated as tryptophane. Without hydrolysis, the values were 3.2 and 3.6 per cent, after alkaline hydrolysis 8.7 and 9.6 per cent, respectively, for treated and control preparations.
9 Based on readings of an atypical purple color. I] Under conditions patterned after a technique of toxoid formation (34), that is,
upon treatment with 0.25 per cent formaldehyde in borate buffer, pH 8 to 9, for 2 weeks at 40”, the apparent tryptophane content of chymotrypsinogen was reduced to 2.0 per cent.
f[ When formaldehyde was added to the digest (1.2 per cent of the protein), only 4.2 per cent tryptophane was found.
Mode of Combination of Formaldehyde-Earlier investigators (7) have shown that the stable reaction product of tryptophane with formaldehyde is a cyclic compound (2,3,4,5-tetrahydrocarboline-4-carboxylic acid), the formaldehyde creating a met,hylene bridge between the amino group and the a-carbon atom of the indole nucleus. It remained to be determined whether formaldehyde acted on tryptophane in peptide linkage in a similar manner, yielding acyltetrahydrocarbolinecarboxylic acid derivatives, or whether the aldehyde was bound in some other manner, the most likely being an addition, as a methyl01 (hydroxymethyl) group, to the indole
TABLE V Reaction of Tryptophane Residues of Tobacco Mosaic Virus with Formaldehyde
Reaction conditions*
per cent 2 1.4
Control
Phosphate None
per cent 2.7 2.4 3.1
ger cent gcr cent per cent per cent 7.0 0.65 6.3 2.7 2.6
* The experiments at neutrality were patterned according to Ross and Stanley (35): 3 per cent protein in 0.1 M, pH 7, phosphate buffer. To obtain the alkaline medium 0.5 ml. of N NaOH was added to 3.5 ml. of this virus solution, then water (1.5 ml.) to give a final protein concentration of 1.9 per cent. Reaction was per- mitted to proceed for 20 hours at room temperature.
t Calculated as tyrosine.
I Analytical s
Phenol + indolet Trvptophaw
$ Both untreated tobacco mosaic virus and that treated with formaldehyde at pH 7.0 had to be heat-denatpred before enzyme digestion could proceed to such an extent that there was maximum release of Folin chromogenic activity. This was not necessary for chymotrypsinogen, serum albumin, and insulin. With regard to lysozyme, see foot-note 3.
0 Based on an atypical purple color.
nucleus. The properties of the formaldehyde derivatives of gramicidin clearly indicate the formation of methyl01 groups. Thus, elementary analyses agree with the methyl01 but not with the methylene formulation (Table VI). Further, quantitative acetylation (29) indicates the appear- ance of about one new hydroxyl group for each equivalent of formaldehyde combining with each tryptophane residue (Table III). The number of hydroxyl groups introduced can consequently be used as an additional
indicating actual combination of these residues with formaldehyde. The observation of Ross and Stanley that simple indoles react with formaldehyde at pH 7 is confirmed in the present study.
means of ascertaining the rate of, and the conditions favoring, the gramici- din-formaldehyde reaction (Table III). The introduction of methyl01 groups into gramicidin is in line with the increased water solubility of the formaldehyde derivative (Table III) (16).
Further confirmatory evidence for the methylol configuration is derived from the comparative lability of the linkage. Treatment of methyl01 gramicidin with hot aqueous sulfuric acid, either under the conditions of hydrolysis and distillation used to determine reversibly bound formalde- hyde or of the chromotropic acid method (19), liberated one-fifth to one- third of the formaldehyde bound by gramicidm. That the liberated formaldehyde was not merely adsorbed was indicated by the constancy of
TABLE VI
Composition of Gramicidin and Methyl01 Gramicidin*
I c
gcr cent
Gramicidin$.................................. 62.8 Gramicidin-formaldehyde product. 61.2 Calculated for methyl01
“ “ methylene 1: $ ,.__._.._.___ 61.5
5 _.... ._ 63.4 -
H N
per cent per cent
7.44 14.5 7.40 13.6 7.40 13.7 7.62 14.1
Hydmyl groups per
10’ gm.t
6 25 24
6
* Averages of repeated analyses on many preparations, on a dry basis. t By quantitative acetylation (29). Model substances gave values of 90 to 95
per cent of the theoretical. $ From the known amino acid composition of gramicidin, Synge (36) calculates
that 144 C atoms should be present for 30 N atoms. The data of Hotchkiss and Dubos (13), Tishler (37), and the analyses recorded here indicate 148, 153, and 152 C atoms for each 30 N atoms, respectively.
8 Based upon the assumption that gramicidin contains 40 per cent tryptophane (14, 16, 36) (19.6 residues per lo4 gm.) and that each typtophane residue adds one methyl01 (or methylene) group.
this value for many different preparations, including both extensively heated and repeatedly recrystallized samples (1). Reactive phenolic methyl01 compounds, such as 2,6-dimethylol p-cresol, are known to release some formaldehyde under similar conditions (38). Model experiments showed that under the distillation conditions used with gramicidin methyl01 skatole and methyl01 2,3-dimethylindole released 0.20 and 0.53 equivalents of formaldehyde, respectively.
More striking is the effect of sodium hydroxide on methyl01 gramicidin. This agent seems to split off the methyl01 group quantitatively. Thus, alkali-treated methyl01 gramicidin shows the full chromogenic value of the original gramicidin. Its hydroxyl groups are reduced to the number
occurring in the original gramiciclm, and it corresponds in elementary analyses closely to a control sample of gramicidin treated with alkali and isolated in the same manner (Table VII). Skatole and 2,3-dimethyl- indole could be regenerated from their respective methyl01 derivatives under the same conditions. The chromogenic activity of other simple indoles, abolished by reaction with formaldehyde, is also largely restored
TABLE VII
Effect of Alkali on Methylal Gramicidin
Conditions of treatments* Analytical results
Sodium hydroxide concentration
Methyl01 gramicidin
Gramicidin
N
None 5 5 5 1 0.1
None 5
Temper- ature
“C.
78
4ott 23 23 23
40
-
-
Tryptophane content
Unhydro- Acid- lyzedt hydrolyzed:
ger cent
<lOTI
37-47 35-45 27
<141j <SlI 35-45
pe* cent (<7.5)7**
34.5 36.2
38.1 14.5 34.1 14.2$$
-
N
per cent
13.6
14.2$$
I Hydmxyl :roups per 10’ pm.5
25
6
6
* In aqueous suspension. Reaction period, 1 day. When high temperatures were used, insoluble products were obtained.
t Approximate values. The curve for unhydrolyxed gramicidin does not coincide with the standard curve for tryptophane.
$ By a mixture of glacial acetic acid and 6 N hydrochloric acid (0.8:3) 18 hours at 100’.
Q Calculated on the basis of the original gramicidin. 7 Based on the atypical purple color. ** Insoluble humin (containing 25 per cent of the total nitrogen) separated during
hydrolysis. tt This preparation contained 55 per cent of the hemolytic activity of gramicidin,
compared to the value of 10 to 20 per cent found with numerous preparations of methyl01 gramicidin.
$$ These alkali-treated preparations of gramicidin and methyl01 gramicidin also had identical carbon (61.4, 61.4 per cent) and hydrogen (7.3, 7.3 per cent) contents.
by the action of sodium hydroxide (Table VIII). It is not surprising that 1 ,&dimethylmdole forms an exception, since this compound was found to yield a 2-methylene rather than a methyl01 derivative.
The tryptophane in formaldehyde-treated proteins was found to resemble that of methyl01 gramicidin in regaining much of its chromogenic value upon treatment with sodium hydroxide (Tables IV and V). This may be
taken as evidence that protein indole groups, like those of gramicidm, add the formaldehyde as methyl01 groups, an assumption which could not as readily be proved by analytical means for proteins as it could be for gramicidin . 8
Locution of MethyZoZ Groups-The location of the methyl01 groups on the indole nucleus remains to be discussed. As previously mentioned, 3- substitut’ed indoles lose, upon reaction with formaldehyde, their chromo- genic activity with the p-dimethylaminobenzaldehyde reagent. The same is the case with 1,3dimethylindole (Table VIII). These observations appeared to provide evidence favoring addition of the formaldehyde to
TABLE VIII Chromogenic Values of Indoles and Formaldehyde Derivatives by Horn-Jones (2.2)
* 2,3-Dimethylindole and carbazole (2,3-phenyleneindole) gave no color at the usual time of reading and a slow development of a bluish color later.
t Treatment with 5 N sodium hydroxide at 53“ for 18 hours regenerated chromo- genic activity from methyl01 skatole but not from methylene bis(l,3-dimethylindole) (cf. Table VII).
the Zcarbon atom, since all indoles with free 2 positions, and only these, give this typical intense blue color.Q
Further evidence for the location of the methyl01 groups was sought on a more strictly chemical basis than was afforded by calorimetry. The fact
8 At elevated temperatures (70“), formaldehyde caused marked losses in the trypto- phane chromogenic activities of proteins even at neutrality, apparently more readily than is the case for gramicidin under such conditions. This has been tentatively attributed to the formation of methylene bridges between aminomethylol groups and indole rings. This reaction will be discussed in a subsequent publication in conjunction with similar reactions involving other cyclic amino acid residues.
9 However, a slow development of a purple-blue color was observed with carbazole and 2,3-dimethylindole.
that skatole and indoleacetic acid and its homologues bind up to 1 equivalent of formaldehyde agrees with the combination of the aldehyde with eit,her the nitrogen or the a-carbon atom, without favoring either position (Tables II and IX). It serves to exclude the side chain of trypto- phane from playing a role in the reaction. The properties of our methyl01 skatole, however, differ significantly from those ascribed by Plant and Tomlinson (39) to 2-methyl01 skatole. Particularly, the lability of the skatole-formaldehyde reaction product in alkali differentiates it from the known hydroxymethylindoles and favors its formulation as an N-methyl01 compound.
TABLE IX Reaction of Simple Indole Derivatives with Formaldehyde*
* 1 mru of compound was allowed to react with 4 to 8 mM of formaldehyde in 10 ml. total volume for 2 days at 53”. The alkaline reaction mixtures contained 0.03 N sodium hydroxide; acid mixtures, 0.1 N acetic acid.
t The reaction product crystallized and was identified as methylene bis(l,3- dimethylindole). Thus, the reaction had proceeded to about 80 per cent completion.
A comparison of the reactivity of 1,3- and 2,3-dimethylindoles towards formaldehyde should demonstrate unequivocally the mode of attachment of the methyl01 group. The results obtained with these model indoles were, however, not quite conclusive (Tables II and IX). Both at room temperature and at 53”, 2,&dimethylmdole bound about 0.5 equivalent of formaldehyde in alkaline and acid media. The reaction product was separated from unchanged 2,&dimethylindole and found to correspond to methyl01 dimethylindole in its composition. In contrast, 1,3-dimethyl- indole did not react appreciably in alkaline solution; in acid solution a crystalline product which separated proved to be methylene bis(l,3- dimethylindole) .l” (1,2-Dimethylindole-3-acetic acid bound no appreci-
10 A small amount of a crystalline product (less than 10 per cent) could also be isolated from alkaline reaction mixtures. Its nitrogen content (9.2 per cent) cor-
able amounts of formaldehyde in either alkaline or acid solution.) Thus, it appears that, in alkaline solution, only 2 ,&dimethylindole combines with formaldehyde, although the reaction does not proceed as readily and completely as it does with indoles substituted only in the 3 position. The properties of methyl01 skatole and the behavior of the dimethylindoles thus favor the attachment of the formaldehyde to the nitrogen, while the calorimetric evidence pointed towards an involvement of the 2 position of the indole nucleus. By analogy with the behavior of phenols and with other indole reactions, one may assume that the formaldehyde primarily reacts with the nitrogen but then may rapidly migrate to the 2 position if this position is unsubstituted. However, it is equally possible that the migration of the methyl01 group to position 2 occurs only during the calorimetric test, which is always performed in hydrochloric acid. This possibility is supported by the observation that both methyl01 gramicidin and methyl01 skatole undergo immediate reactions when treated with hydrochloric acid at room temperature or at 0” in glacial acetic acid solu- tion. Both methyl01 products are transformed into yellow to red-brown precipitates, while unmodified gramicidin and skatole are unaffected by similar treatment. The nitrogen content of neither methyl01 compound is altered, but their chromogenic activities can no longer be regenerated by treatment with alkali. Thus, while it has not been possible to crystallize the acid-treated methyl01 skatole or to identifiy it with Plant and Tomlin- son’s (39) 2-methyl01 skatole, indications are that the acid conditions of the test cause the change responsible for the loss in chromogenic activity of formaldehyde-treated indoles. Thus, the evidence favors the attach- ment of the methyl01 group to the nitrogen as indicated by formula I.”
Of two other attempted approaches to the problem, one has given no answer and the other favored the above conclusion. Acetic anhydride treatment of gramicidin in glacial acetic acid (in contrast to pyridine) has been found to yield a product containing considerably more acetyl groups than correspond to its hydroxyl content, which thus have been allocated to the only other polar groups, that is, the indole groups (16). Since this product had the full chromogenic value of gramicidin and gave off acetic
responded to methylene bis(l,3-dimethylindole); yet it differed from this product,, as formed in acid media, by a slightly higher melting point, (158”). The mixed melting point between the two showed a marked depression (125-130”).
l1 Other gramicidin derivatives carrying substituents on the indole nitrogen, notably the ‘sulfamate and the acylated products, show the chromogenic value cor- responding to their original tryptophane contents (16). The different behavior of these and the formaldehyde derivative is understandable in view of the known reversibility, by acid hydrolysis, of all but the formaldehyde fixation. Thus the sulfate group or various acyl groups tend to be split, off rather than to migrate to the 2 position.
acid upon acid hydrolysis, it was regarded as a 1-acetyl derivative.ll It was hoped that formaldehyde treatment of this material might throw light on the mechanism of the formaldehyde reaction. However, no conclusions could be drawn from the observed ability of this derivative to react with formaldehyde, since the acetyl groups were found to be largely split off during the reaction with formaldehyde in alkaline solution.
On the other hand, acetylation (in glacial acetic acid) of methyl01 grami- cidin introduced acetyl groups corresponding in number to the hydroxyl groups (25 per lo4 gm.), but introduced no additional amount such as might have been expected if acetylation of the indole nitrogen had occurred (16). This may be regarded as additional evidence that the methyl01 groups are located in the 1 position.
Preparative
Methylol Skatole-To 786 mg. (6 mM) of skatole, dissolved in 40 ml. of ethanol, were added 2.25 ml. of N sodium hydroxide and 30 ml. of 3.85 per cent aqueous formaldehyde. After 6 days at room temperature the mixture was diluted with much water, extracted twice with ether, and the ether washed with water until no more formaldehyde could be detected in the washings. The ether was dried and evaporated. The residue (857 mg., crystals and oil) was extracted with cold petroleum ether, then dissolved in benzene, and petroleum ether added. Crystals separated from both solutions (360 and 260 mg.), melting point 52” after recrystalliza- tion from benzene and petroleum ether. In contrast to skatole, this compound gave no color with tryptophane reagents (Table VIII).
C,oH1lON. Calculated. C 74.5, H 6.9, N 8.7 Found. <‘ 74.5, Lc 7.0, (‘ 8.7
When 55 mg. of methyl01 skatole were suspended in 1 ml. of 5 N sodium hydroxide and heated to 53” for 2 hours, the crystals turned into an oil, then crystallized. The product was identified as skatole by melting point and mixed melting point.
Methylene B&(1 , .!Mimethylindole)-A solution of 1 ml. of 1,3-dimethyl- indole in 7.5 ml. of alcohol was mixed with 2 ml. of 38.5 per cent aqueous formaldehyde, and 0.5 ml. of 3 M acetic acid and held at room temperature for 7 days. The deep red solution deposited crystals which were isolated after cooling, and washed with cold alcohol. Yield, 540 mg. Recrys- tallization from alcohol yielded colorless crystals, m. p. 138-148”.
C2&.$J2. Calculated. C 83.5, H 7.3, N 9.3, mol. wt. 302 Found. “ 83.2, “ 7.3, “ 9.2, “ “ (Rast) 327, 314
The same compound but in somewhat smaller yield (303 mg.) was obtained after a similar mixture had been heated at 78” for 2 hours.
Methyl01 2, S-Dimethylindole-725 mg. (5 mM) of 2,3-dimethylindole were treated with formaldehyde as was skatole. After 4 days at room temperature, a 1 ml. sample was diluted for formaldehyde analysis (which indicated that 0.49 equivalent had been bound by the indole). The rest of the solution was worked up as above. From the residue (743 mg.), unchanged starting material was extracted with boiling petroleum ether (b. p. 30-60”). The residue from this operation (340 mg.), after recrystal- lization from benzene-petroleum ether, melted at 98-100”.
CL1H130N. Calculated. C 75.5, II 7.4, N 8.0 Found. “ 75.5, “ 7.5, ‘( 7.9
Treatment of 38 mg. of methyl01 2,3-dimethylindole with 5 N sodium hydroxide at 53” for 20 hours, yielded 29 mg. of 2,3-dimethylindole, char- acterized by melting point, mixed melting point, and nitrogen analysis (9.7 per cent,).
Comments
The present investigation has demonstrated that formaldehyde reacts with gramicidin only on the tryptophane residues. The fact that the toxicity is thereby decreased, whereas the antibacterial activity is retained (15, 16), suggests that tryptophane residues in other biologically active compounds may also play an important role in mediating their activities. For.example, the mode of detoxication of toxins with formaldehyde to form toxoids has often been the subject of speculation. The work of Farrell (34), as confirmed by Dubos and Geiger (40), with Shiga toxin suggests that the reaction of formaldehyde may be that of combining with part of the indole residues. This worker found that incubation of the crude toxin with 0.5 per cent formaldehyde failed to detoxify the material even after 18 months at 37“. By adjustment of pH, he observed a reduc- tion of toxicity to one-seventh at pH 6.7 and to one-fiftieth at pH 8.2, without loss of the antigenic activity. The maximum loss of toxicity and retention of antigenicity were observed at pH 8.5 aft,er 2 weeks at 37”. Under similar conditions, the tryptophane residues of chymotrypsinogen reacted to about 67 per cent (Table IV, 11 foot-note). Although various other polar groups react with formaldehyde under such conditions, none of these reactions is known to be as dependent upon an alkaline pH as is that of the tryptophane residues. Thus, a reaction mechanism analogous to that of gramicidin may be the basis of the formation of some formalm toxoids, as was suggested by Velluz (9).
Any generalizations, however, would appear to be premature in view of the effectiveness of ketene (41) and of acetic anhydride in pyridine (42) for the preparation of some toxoids and vaccines. These acetylating agents
and conditions may not affect the indole groups of gramicidin,lz nor have they been shown to act on these groups in any typical protein.
Thus, there are indications that the desirable effects (reduction of toxicity without loss of antigenicity) may be produced by modifying the indole groups in some and the amino, or possibly both types of groups, in other toxic proteins. However, the tobacco mosaic virus protein, probably the most exhaustively studied in this regard, represents further problems, inasmuch as it is not inactivated by acylation of its amino groups (43), but is inactivated by formaldehyde (35, 44) under conditions in which few if any of its indole groups appear to react.
It is of interest that “hydroxyalkylation” has been shown to reduce the toxicity of several pharmacologically active synthetic compounds (45).
SUMMARY
Gramicidin binds rapidly in alkaline solution an amount of formaldehyde equivalent to its tryptophane content. In acid solution the reaction proceeds only at high temperature and formaldehyde concentrations.
The formaldehyde adds as methyl01 groups probably to the nitrogen of the indole rings. Formaldehyde is released only partially during acid hydrolysis, but completely and without damage to the gramicidin molecule in strong alkali.
The chromogenic activity of the tryptophane residues is largely abolished through combination with formaldehyde and regenerated by strong alkali.
The tryptophane residues of proteins appear to react with formaldehyde under the same conditions and in a manner similar to those of gramicidin At room temperature there is little reaction at pH 7 to 8, even at high formaldehyde concentration, but at pH 11 the reaction is completed rapidly, even at low formaldehyde concentration.
All 3-substituted indole derivatives, including proteins rich in trypto- phane, bind formaldehyde in boiling N to 20 N sulfuric acid.
Simple 3-substituted indoles bind up to 1 mole of formaldehyde at room temperature, both in alkaline and in acid solution. 2,3-Dimethylindole reacts incompletely, even at elevated temperature. 1 ,3-Dimethylindole reacts appreciably only in acid yielding methylene bis (1,3-dimethylindole) . Pure methyl01 derivatives were obtained from skatole and 2,3-di-
I* Experiments to introduce acetyl groups on the indole residues of gramicidin with ketene were ambiguous. A vigorous stream of ketene was passed into a suspen- sion of gramicidin in a 1 M solution of sodium acetate for 30 minutes (at 0’). The solution was kept slightly alkaline by the simultaneous addition of 1 M sodium hydroxide. The isolated gramicidin was found to have an acetyl content correspond- ing only to its hydroxyl groups (six per 10’ pm.) but not to its indole content (19.6 per IO4 pm.). Somewhat more acetyl was introduced when gramicidin was extensively ketenized in alcoholic solution.
methylmdole. 1,2-Dimethylmdole-3-acetic acid does not react with form- aldehyde.
The authors are indebted to L. M. White and G. Secor for numerous elementary analyses, to J. C. Lewis for the assays for antibacterial activity, to K. P. Dimick for the assays for hemolytic activity, to B. G. Edwards for determinations of solubility, and to C. Cleaver, S. Ahnger, and E. D. Ducay for valuable technical assistance.
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