improved chemical synthesis and enzymatic assay ofΔ1-pyrroline-5-car☐ylic acid

ANALYTICAL BIOCHEMISTRY 64, 8 5 - 9 7 (1975)

Improved Chemical Synthesis and Enzymatic Assay of N -Pyrroline-5-Carboxylic Acida

IRENE WILLIAMS AND LEONARD FRANK

Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland

Received July 8, 1974: accepted September 6, 1974

A~-Pyrroline-5-carboxylic acid, an intermediate in both the biosynthesis and degradation of L-proline, has been synthesized by the periodate oxidation of hydroxylysine and isolated as a pure compound, as indicated by enzymatic assay with pyrroline-5-carboxylate reductase from Escherichia coli. Some features of the instability in solution of 2x~-pyrroline-5-carboxylic acid have been studied, leading to the conclusion that the rate of decomposition is sensitive to concentration of the compound. Colorimetric assay with o-aminobenzaldehyde was found to be an inadequate measure of the pyrroline compound in partially decomposed solutions.

In a wide range of spec ies , A l - p y r r o l i n e - 5 - c a r b o x y l i c ac id (P5C) 2 is an i n t e rmed ia t e both in the b io syn the s i s and ae rob ic c a t a b o l i s m of L-prol ine (1)_ T h e c o m p o u n d was first r e cogn i zed as a me tabo l i c i n t e r m e d i a t e by Voge l and Dav i s (2) who also d e s c r i b e d its chemica l syn thes i s and its r eac t iv i ty with o - a m i n o b e n z a l d e h y d e (o-AB)_ This reac t ion , fo l lowing S t r e c k e r (3), has been wide ly used as a quan t i t a t ive a s s a y for P5C.

A l t h o u g h G o o d and Mi tche l l (4) d e s c r i b e d a syn thes i s o f P5C as its d i e thy l ace t a l , all w o r k e r s requi r ing the c o m p o u n d have e m p l o y e d the Voge l and Dav i s procedure_ In this p r o c e d u r e , s u b s e q u e n t l y ref ined by S t r e c k e r (3), g lu tamic s e m i a l d e h y d e was gene ra t ed by ac id h y d r o l y s i s o f y, y - d i c a r b e t h o x y - y - a c e t a m i d o b u t y r a l d e h y d e . T h e s e m i a l d e h y d e ex i s t s in so lu t ion p r imar i ly as its cyc l ic a ld imine , P5C (2). S t r e c k e r (3) pur i f ied P 5 C by c h r o m a t o g r a p h y on D o w e x 50: e lu t ion with HCI y i e l d e d the h y d r o c h l o r i d e as a solid. Whi le this sol id cou ld be s tored , its pur i ty was on ly 80% by e n z y m a t i c a s say with P5C d e h y d r o g e n a s e . S t r e c k e r found , as d id Voge l and Davis , tha t P5C is r a the r uns t ab le in solut ion.

A need for f r equen t p r e p a r a t i o n o f P 5 C led us (5) to e x a m i n e a f a s t e r and s imple r syn the t i c route : the p e r i o d a t e ox ida t i on o f 6 - h y d r o x y l y s i n e .

Supported in part by Public Health Service Grant AI-08470 flora the National Insti- tute of Allergy and Infectious Diseases, National Institutes of Health and by the General Research Support Grant to the University of Maryland School of Medicine.

Abbreviations used: P5C, A~-pyrroline-5-carboxylic acid; o-AB, o-aminobenzaldehyde.

85

Copyright c~ 1975 by Academic Press, Inc. Printed in the United States. All rights of reproduction in any form reserved.

86 W I L L I A M S A N D F R A N K

From the known reactivities of periodate one would predict that the products of its action on hydroxylysine are ammonia, formaldehyde, and glutamic semialdehyde. In fact, this reaction was initially employed by Van Slyke et al. (6) for the quantitative determination of hydroxylysine by measuring ammonia released.

The present report describes both the preparative conditions for this reaction, which lead to a high yield of apparently pure P5C, and a simple enzymatic assay for P5C.

MATERIALS AND METHODS

Special chemicals. 8-Hydroxylysine monohydrochloride (a mixture of hydroxy-Dk-lysine and allohydroxy-Dk-lysine) was purchased from Cal- biochem. According to the supplier, the compound was chroma- tographically homogeneous; we did not investigate its purity. L- [U-14C]- proline was obtained from New England Nuclear Corp. N A D P H was purchased from the Sigma Chemical Co. and ninhydrin from Pierce Chemical Co. Nutrient broth was obtained from Consolidated Labora- tories, o-AB was purchased from K&K Laboratories and stored at -20°C. Solutions of the reagent were stored in brown bottles at 4°C; they were stable for several weeks. A sample of o-AB from Pfaltz and Bauer yielded turbid solutions. However, this reagent solution could be clarified by centrifugation and, when used in the assay, it gave results identical to those obtained with the former material. Dowex 50 (200-400 mesh, 4% cross-linked) ion-exchange resin was purchased from BioRad Laboratories under their designation, AG50W. DEAE was Whatman's product, DE-23. The resin was prepared according to pro- tocols supplied by the Company and equilibrated with 10 mM Tris-C1 buffer (pH 7.5) before pouring the column.

Total nitrogen was determined by a Kjeldahl procedure essentially as described by Hiller et aL (7).

Qtlalitative detection of P5C or proline. A 20 /zl sample of column effluent was mixed with 20 /zl of 3 M sodium acetate and 500 /~1 of 0.15% ninhydrin in glacial acetic acid in a small test tube. The tube was held in a rack on a hot plate; when 0.5/zg or more of P5C or proline was present, a cherry-red color appeared shortly before the solution boiled.

Quantitative assay of P5C with ninhydrin. Aliquots of solution containing P5C were mixed with a HC1 solution to give a final concentration of 1 N HC1. Appropriate dilutions of the resulting solution were prepared using a diluent containing the P5C solvent (e.g., Tris buffer), similarly mixed with HC1. To 0.3 ml of such samples were added 0.3 ml of 3 M sodium acetate and 4.0 ml of 0.15% ninhydrin in glacial acetic acid (8). The tubes were capped and incubated at 50°C for 15 rain and then cooled to room temperature; the absorbance at 535 nm was a linear

A 1-pYRROLINE- 5-CARBOXYLIC ACID 87

function of P5C concentrat ion with a molar absorpt ion coefficient of 4600 M - l c m - L

Quantitative assay of P5C with o-AB. Aliquots of solution containing P5C were mixed with a HCI solution to give a final concentrat ion of 1 N HCI; 2 ml of the resulting solution were mixed with 1.0 ml of 0.5% o-AB in 100% ethanol. After 40 min at room tempera ture the solutions were read at 444 nm against an exact blank. Standardizat ion of this assay, dis- cussed in Results, yielded a molar absorpt ion coefficient of 2940 M -1 c m - L Virtually identical absorbance measurements were obtained in a Beckman model D B - G spec t rophotometer and in a Gilford model 300-N microsample spec t rophotometer equipped with a cuvette of 10 mm light path.

Preparation of L-['4C]-P5C_ A 2 ml reaction mixture, pH 8.7, containing 15/xmoles of L-[ '4C]-proline, 200/xmoles of Tr i s -Cl , 40/ , tmoles of MgC12, and 1.2 units of proline oxidase (see below) was incubated with shaking at 37°C for 60 rain. One milliliter of 15% trichloracetic acid was added and the denatured protein was removed by centrifugation. L-[14C]-P5C was isolated by chromatography of the supernatant on Dowex 50, essentially as described in Results tbr chemically synthesized P5C_ Approximate ly 6/xmoles of [ ' 4C] -P5C were obtained.

Assay of/)rolitle oxidase. In the absence of pyridine nucleotide, the proline oxidase complex from Escherichia coli catalyzes the oxidation of l,-proline to t_-P5C (9). Accumulat ion of P5C was followed by reaction with o-AB; 1 ml reaction mixtures, containing 100 ~moles T r i s -C l buffer (pH 8.7), 20 /~moles MgCI2, 10 p, moles I,-proline, and enzyme were incubated at 37°C with shaking for 10 rain. After addition of 1 ml 10% trichloracetic acid and 1 ml of 0.5% o-AB (in absolute ethanol), the reaction mixtures were allowed to stand at room tempera ture for 40 rain, the denatured protein was then removed by centrifugation and the absorbance at 444 nm of the supernatants was determined. The production of 1 ~mole of P5C in the 1 ml incubation mixture resulted in an absorbance of 0.98. The amount of P5C produced was a linear function of enzyme concentrat ion in the range 0 -2 /xmoles P5C_ One unit of proline oxidase activity was defined as the quantity of enzyme catalyzing the product ion of 1 /zmole of P5C/min.

Assay of P5C reductase. The enzyme preparat ion described below catalyzes the reduction of L-P5C to L-proline with either N A D P H or N A D H , but the former is most convenient to use because of interfering N A D H oxidase activity. Progress of the reaction was followed by recording the decrease in absorbance at 340 nm as a function of time, and at room temperature. The reaction was started by adding 100 /xl of enzyme (or a suitable dilution) to a reaction mixture of 900/~l containing 0 .3 /xmole of N A D P H and 0.8 ~mole of DL-P5C. The mixture was but'-

88 WILLIAMS AND FRANK

fered at pH 8 by Tris-C1 at a final concentrat ion of 0.2 M. One unit of P5C reductase activity was defined as the quantity of enzyme catalyzing the oxidation of 1 /zmole of N A D P H / m i n .

Preparation of proline oxidase. Proline oxidase was obtained from a double mutant (9) of E. coli but the procedure given is equally applicable to wild-type cells of Strain B (A.T.C.C. no. 11303). The bacteria were grown at 37°C with vigorous aeration in a medium consisting of B7 salts (10) supplemented with 20 mM K acetate, 15 mM NH4C1, and 10 mM L- proline. An inoculum for 4 liters of medium was prepared by several successive growth cycles of the bacteria in the same medium. The ceils were allowed to increase 300-fold from an initial density of 2 × 106 cells/ml. They were harvested by centrifugation and washed in 20 mM K phosphate, pH 7.0. Following resuspension in wash buffer to a density of 150 mg wet wt of cells/ml, the cells were disrupted by passage through a French pressure cell under 28,000 psi; unbroken cells and debris were removed from the extract by centrifugation at 34,000g for 30 min. All operations following cell disruption were carried out at 5°C. Sixty milliliters of the centrifuged extract was dialyzed for 2 hr against wash buffer and then centrifuged for 2 hr at 100,000g to sediment the proline oxidase activity. The pellets were resuspended in 20 ml of wash buffer and further homogenized by passage through a 25-gauge needle. Under the assay conditions described above, this preparation contained 1.5 units of proline oxidase/ml. After storage at - 9 5 ° C for 9 mo, the enzyme activity had decreased by 40%.

Preparation of L-P5C reductase. L-P5C reductase was obtained from E. coli, Strain B. Two liters of nutrient broth was inoculated with a broth culture of the cells to an initial density of 2 x 106 cells/ml. After 15 hr of growth at 37°C with vigorous aeration, the cells were harvested by centrifugation, washed in 10 mM Tr is -Cl (pH 7.5), resuspended in the wash buffer to a density of 150 mg wet wt of cells/ml, and disrupted as described above for the proline oxidase preparation. The extract was centrifuged at 34,000g for 30 min and the resulting supernatant was further centrifuged at 100,000g for 2 hr. Twenty-four milliliters of this supernatant was loaded on a 2 x 35 cm column of D E A E (see Special Chemicals, above) and the following sequence of buffers was passed through the column: 50 ml 10 mM Tris-C1 (pH 7.5) ( " B U F F E R " ) ; 100 ml B U F F E R containing 0.1 M Kcl; 100 ml B U F F E R containing 0.2 M KC1; 50 ml B U F F E R containing 0.3 M KC1. At this point, the collection of 4 ml fractions was initiated and a further 50 ml of the same buffer was passed through the column. Finally, B U F F E R containing 0.35 M KC1 was applied to the column. The enzyme activity began emerging in the last fraction collected during elution with B U F F E R + 0.3 M KC1. Appropriate fractions were pooled, giving 50-60 ml of an enzyme preparation only eightfold purified but virtually lacking an interfering

A I -pYRROLINE- 5 -CARBOXYLIC ACID 89

N A D P H oxidase activity. The yield of e n z y m e was 7 0 - 8 0 % of the total P5C reductase activity in the extract. Under the assay condit ions described above, the pool of e n z y m e contained approximately 20 units o f P5C reductase/ml. Whether or not the e n z y m e preparation was supplemented with 10 mg/ml of serum albumin it retained 50% of its activity during storage at - 9 5 ° C for 4 mo.

RESULTS

S y n t h e s i s a n d i s o l a t i o n o f D L - P 5 C . T w o mill imoles o f hydroxylys ine was dissolved in 28 ml of water in a brown bottle and the solution was brought to 4°C. A 50 mM solution of sodium metaperiodate, prepared in dim light, was adjusted (glass electrode) to pH 7.0 with a small vo lume of 1 N N a O H : after temperature equilibration in ice, 44 ml of the neutralized periodate solution was added quickly to the hydroxylys ine solution. Eight minutes after mixing the two solutions, remaining periodate was destroyed with 0.7 ml of 1 M glycerol. After 2 min the reaction mixture, held in ice, was acidified with 0.6 ml of 6 N HCI. Enzymat ic assay with P5C reductase (see below) indicated a 94% yield o f P5C, based on hydroxylysine.

The entire reaction mixture was al lowed to flow into a 2 x 62 cm col-

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FIG. 1. Chromatographic isolation of P5C on D o w e x 50 The column was eluted with I

N HCI at room temperature after application of the sample at 4°C, as described in the text. P5C concent la t ion in the fractions (8 5 ml) was est imated by o - A B assay (see Materials and Methods) and relative N a + concentrat ion was est imated with a sodium electrode ( C o m i n g ) after neutralization of the effluent with Tris, free base.


umn of Dowex 50 equilibrated with water (after thorough washing with 1 N HCI), and at 4°C. Sample adhering to the walls of the column was washed into the resin with 20 ml of 0.05 N HC1 and elution with 1.0 Y HCI was then initiated at a flow rate of 50 ml/hr. Usually, the column was removed to room tempera ture for the remainder of the procedure.

Under these conditions ammonia, unreacted hydroxylysine, and a degradation product of P5C were retained more strongly than P5C while formaldehyde and iodate emerged immediately. Sodium ion was eluted before, and well separated from, P5C (Fig. 1). P5C was detected in the effluent (see Materials and Methods) and appropriate fractions were pooled, yielding an approximate ly 9 mM solution of PSC. The compound was stable at 4°C for at least l0 mo in this HCI solution. However , all a t tempts to obtain a pure solid failed. Although, as indicated above, P5C was generated in 94% yield from hydroxylysine the yield of isolated P5C was lower; it varied from 77 to 87% in 13 independent preparations.

Calibration of the reaction of P5C with o-AB. On the assumption that the solution of P5C did not contain non-nitrogenous compounds which react with o-AB, we determined a color yield for the P 5 C - o - A B reaction based on total nitrogen in the P5C solution. Under the conditions described in Materials and Methods a range of aliquots from each of eight independent preparat ions of the P5C solution in 1 N HC1 were assayed and the pooled results were analyzed as the regression of absorbance on micromoles of nitrogen (Fig. 2). The calculated regression line

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FrG. 2. Calibration of the reaction of P5C with o-AB_ Eight independent preparations of P5C were standardized according to total-N and multiple aliquots f rom each were assayed with o-AB under conditions given in Materials and Methods . The regression line was calculated by the method of Leas t Squares, using all of the data points. Spectrophotome- tric measu remen t s were made in a Gilford Microsample Spect rophotometer equipped with a cuvet te of 10 m m light path.

AI-PYRROLINE-5-CARBOXYLIC ACID 91

(least squares) for the pooled results had a slope of 0.981 OD//xmole N/3 ml reaction mixture; the standard error of this slope was 0.009 and the 95% confidence limits were 0.981 + 0.023. The molar absorpt ion coefficient derived from these results, 2940 M-lcm -1, is 9% greater than the most recent one reported by Strecker (11) for the o - A B - P 5 C reaction. We determined that the difference was not due to the somewhat different assay conditions used in our laboratories; it seems likely that it reflects a higher level of purity of our P5C. This conclusion, of course, assumes that our solutions did not contain nitrogenous impurities which react with o-AB to give a higher color yield than P5C.

The relatively small standard error of the pooled regression coefficient (Fig. 2), together with visual inspection of the data, suggests that the synthetic procedure for P5C, while differing in yield f rom run to run, yields a highly reproducible preparat ion of isolated P5C. This conclusion is supported by the results of enzymatic assay of the P5C as well_

Enzymatic assay of P5C with P5C reductase; steric specificity of the reaction. The enzyme preparat ion from E. coli (see Materials and Methods) catalyzes the reduction of P5C to proline with N A D P H as reductant. The following two experiments indicated that the enzyme is specific for the L-isomer of P5C.

In the first experiment, DL-P5C was allowed to react with the enzyme and N A D P H until periodic assay with o-AB indicated no further change in the concentrat ion of P5C. It was found that almost exact ly one-half of the o-AB reactivity was lost during this incubation (Fig. 3), a result consistent with the utilization of only one isomer of P5C. For this experiment it was necessary to use a N A D P H - g e n e r a t i n g system rather than a high concentrat ion of N A D P H itself because of the reactivity of re- duced pyridine nucleotides with o-AB (12).

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p-MOLES DL-PSC ADDED

Fro. 3. P5C remaining after treatment of DL-P5C with P5C reductase. The indicated quantities of DL-P5C were incubated with enzyme under conditions similar to those given in the text for enzymatic assay of P5C except that N A D P H was replaced by 0.07 mM N A D P and an NADPH-generat ing system consisting of glucose-6-phosphate and glucose- 6-phosphate dehydrogenase. P5C was measured with o-AB as described in Materials and Methods. The data shown were those obtained after 20 min of incubation at room temperature, a time at which o-AB reactivity had decreased to a constant level.

92 W I L L I A M S A N D FRANK

In the second experiment, the quantitative reduction of the L-isomer of P5C was demonstra ted with the aid of L-[14C]-P5C (see Materials and Methods). L-[14C]-P5C was incubated with P5C reductase under the conditions given below for the enzymatic assay. After a suitable period, trichloracetic acid was added to a final concentrat ion of 5% and the mixture was supplemented with carrier DL-P5C and proline. Follow- ing centrifugation to remove precipitated protein, the supernatant was chromatographed on Dowex 50 (see Isolation of P5C, above). P5C and proline were located in the effluent by reaction with ninhydrin and the total radioactivity was measured in the respective peaks. Less than 1% of the [14C]-P5C remained after the incubation while 96% of the starting quantity of radioactivity was recovered f rom the column as proline.

In addition to establishing the steric specificity of the P5C reductase reaction, these experiments showed that the reaction proceeds to com- pletion. Thus, the change in absorbance at 340 nm could be equated to the amount of P5C present without the internal standardization required of assays dependent upon rate measurements or incomplete reaction.

Conditions for enzymatic assay ofP5C. A solution of P5C in I N HC1 was mixed with water and with a solution of Tris, free base (2 equiv/equiv of HC1) to give a solution which was 0.2 M in Tris, approximately 1 mM in oL-P5C, and at pH 8. Several different-sized aliquots of the neutralized P5C were transferred to small tubes and the volume in each tube was made to 0.8 ml with 0.2 M Tris-C1, pH 8.0; 0.3/~mole of N A D P H (from a stock solution prepared in the Tris buffer) and 2 units of L-P5C reductase were added to make the total volume 1 ml. After mixing, the tubes were incubated at room tempera ture for 10 rain; this was sufficient time for the reaction to reach complet ion at concentrat ions of L-P5C to a maximum of 0.2 mM. The final absorbance at 340 nm was determined. While the change in absorbance (due to the oxidation of N A D P H ) could be obtained by conducting each reaction individually in the spec t rophotometer and taking a zero-t ime reading, we chose an approach more convenient for the processing of multiple samples. For a given group of different-sized aliquots of P5C solution we estimated the zero-t ime absorbance by extrapolating to zero P5C the regression of final O D (optical density) on P5C sample volume. This procedure yielded more consistent results than one in which the zero-time absorbance was est imated by preparing simulated reaction mixtures lacking P5C.

Purity of the P5C by enzymatic assay. Our inability to obtain P5C as an acceptable solid led us to the use of total nitrogen as the basis for as- sessment of chemical purity. Five independent batches of P5C in 1 N HCI were subjected to total nitrogen analysis and neutralized solutions were prepared from each batch, as described above. Multiple aliquots

A 1-pYRROLINE- 5-CARBOXYLIC ACID 93

from each of these solutions were assayed with P5C reductase, as described above. The results of these assays, encompass ing all five preparations of P5C, are shown in Fig. 4, graphed as the regression of absorbance change on quantity of P5C-nitrogen added. The line in Fig. 4 represents the expected result for stoichiometric reduction of L-P5C in a preparat ion of pure DL-P5C. It was drawn with the assumptions that 1 mole of N A D P H is oxidized for each mole of L-P5C present and that the absorption coefficient for N A D P H is 6220 M-lcm -1 (13). The analytical data closely approximate the expectat ion, O D = 3.11/mN DL-P5C-N. Statistical analysis of the pooled data yielded a mean regression coefficient of 3.14-2-_ 0.09 (95% limits) OD/mM N. Taking the expected value of 3.11 as indicative of "' 100%" purity, this result is equiv- alent to an average purity between 98 and 104%.

Stability of P5C. In our experience P5C is a rather labile compound whose rate of decomposi t ion is determined particularly by the interac- tion of solution pH and concentrat ion of P5C_ Unfortunately, destruc- tion of the compound is rapid under precisely those conditions which usually pertain to enzymological and other biological studies.

The 9- 10 mM solution of P5C which results from our method of isola-

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FIG. 4. Purity of P5C by enzymatic assay. As described in the text, different-sized aliquots of a neutralized solution of Dk-P5C were incubated with PSC reductase and N A D P H and the change in absorbance at 340 nm determined in the Gilford Spectropho- tometer. Each point represents the absorbance change in a single incubation containing a known concentration of DI.-P5C N. The data shown were obtained with five independent preparations of P5C. The line in the figure is the expected result for quantitative reduction of exactly one-half of the P5C assuming pure DI.-PSC, stoichiometric reduction of the k- isomer by N A D P H , and a molar absorption coelficient for the latter of 6220 M Jcm ~. The results of statistical analysis of the data are given in the text.


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FIG. 5. Decomposition of P5C as a function of concentration and time: 21 ml of a stock solution of PSC in 1 N HCI was evaporated to dryness in a rotary evaporator and the resi- due of 200 ~moles of P5C was dissolved in 0.1 ! M Tris to give a solution at pH 7.3 and containing 50 mM P5C. This solution was diluted with 0.11 M Tris-Cl (pH 7.3) to give separate solutions containing the different concentrations of P5C shown in the figure. The five solutions were incubated at 37°C and aliquots were removed at the intervals shown. These were assayed with o-AB (see Materials and Methods) after dilution with 1 N HCI to retard further decomposition.

t ion cou ld be e v a p o r a t e d to d r y n e s s wi th l i t t le or no de s t ruc t i on o f P 5 C p r o v i d e d the v o l u m e o f so lu t ion d id not e x c e e d a p p r o x i m a t e l y 40 ml. G r e a t e r vo lumes , e v a p o r a t e d as a single ba tch , y i e lde d var iab le ex t en t s o f d e s t r u c t i o n o f P5C which, h o w e v e r , d id not e x c e e d 1 0 - 1 5 % . D e c o m - pos i t i on was not p r e v e n t e d by conduc t i ng the e v a p o r a t i o n in a n i t rogen a t m o s p h e r e nor by the subs t i tu t ion o f t r i f luorace t ic acid as the so lven t for e lu t ion o f P5C f rom the co lumn. L ikewise , l yoph i l i za t ion o f the HC1 so lu t ion o f P5C, r a the r than r o t a r y e v a p o r a t i o n , d id not p ro t ec t the compound .

W e inves t i ga t ed the labi l i ty o f P 5 C in a n u m b e r o f so lven ts by evap - o ra t ing 2 0 - 4 0 ml o f the s tock so lu t ion in H C I and d i s so lv ing the P 5 C re s idue unde r the de s i r ed cond i t ions . In genera l , r ed i s so lu t ion in w a t e r or in H C I so lu t ions o f 0.2 or 1.0 N y ie lded so lu t ions o f P5C which we re s tab le for 24 hr or more , even at 25°C and with P5C c o n c e n t r a t i o n s nea r 100 mM. But a d j u s t m e n t o f the p H to the neu t ra l range or d i s so lu t ion o f P5C in a va r i e t y o f buffers in the p H 7 - 8 range resu l t ed in uns tab le solu- t ions . T h e effect o f P5C c o n c e n t r a t i o n on its ra te o f de s t ruc t i on in Tr i s

buffer at 37°C is shown in Fig. 5. H e r e , us ing o - A B reac t iv i ty as c r i te - r ion, 50 mM P5C los t 30% o f its r eac t iv i ty in 1 hr while 1 mM P 5 C r e m a i n e d e s sen t i a l l y u n c h a n g e d for 3 hr.

Measttrement of P5C decomposition. Whi le loss o f o - A B reac t i v i t y c l ea r ly signals d e c o m p o s i t i o n o f P5C, resu l t s such as those p r e s e n t e d in Fig. 5 are not su i tab le for k inet ic ana lys i s b e c a u s e d e c o m p o s i t i o n p rod - uct(s) t h e m s e l v e s r eac t with o - A B . Thus , c h r o m a t o g r a p h y on D o w e x 50

A ~-PYRROLINE- 5 -CARBOXYLIC ACID 95

4 0

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NINHYDRIN ASSAY

i i i i i i i i i . . . .

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Fl(;. 6. Decomposi t ion of P5C as a function of time, as est imated by dill 'elent assays. A

solution uf P5C (approx 50 mM) was prepared in 0.05 M Tr i s -Cl (pH 7.5) as descr ibed in the legend to Fig. 5. The solution was incubated at 37°C and aliquots wine removed at the

intervals shown: they were diluted with 1 N HC1 and subsequently assayed for P5C by the three methods indicated in the figure, as described in the text.

of partially decomposed solutions yielded, in addition to PSC, o-AB- reactive material which required more vigorous elution procedures (4 N HCI) than does PSC_

That o-AB reactivity could not be equated with P5C under conditions of decomposi t ion could be inferred, also, f rom enzymatic assay of the same solution: the latter assay yielded est imates of P5C concentrat ion which were lower than those estimated by o-AB assay (Fig. 6). Appro- priate mixture experiments with decomposed and flesh solutions of PSC indicated that the enzymatic assay was reliable even when authentic P5C consti tuted only 20% of all P5C material in the solution.

Ninhydrin assay (see Materials and Methods) yielded est imates of P5C concentrat ion more in accord with those obtained by enzymat ic assay (Fig. 6). The reaction of PSC with ninhydrin was studied in some detail by Strecker (3). Our quantitative ninhydrin assay represents a modification of the method of Piez et al. (8) who showed that proline

96 WILLIAMS AND FRANK

reacts with ninhydrin under acid conditions to produce red-colored solutions at I O0°C and yellow-colored ones at room temperature. We determined that, at 50°C, P5C produced a bright red color while proline, under the same conditions, yielded yellow solutions with absorption maximum at 350 nm.

DISCUSSION

The synthesis of P5C by periodate oxidation of hydroxylysine greatly reduces the time and complexity associated with the previous synthetic methods for this compound. In addition, the isolated P5C appears to be pure, within the limits of error of our enzymatic assay. The method is applicable, in principle, to the preparation of optically specific L- or D-P5C by starting with the appropriate isomer of hydroxylysine.

Our observations on the behavior of P5C in aqueous solution add to the recognition of the lability of this compound by Vogel and Davis (2) and by Stecker (3). Variables affecting decomposition of P5C appear to be solution pH and concentration. Increase in the rate of decomposition with concentration is consistent with the existence of polymerization reactions (2), as is our recognition of o-AB-reactive material which binds strongly to Dowex 50. We should add to the results presented above that P5C is largely decomposed when chromatographed in citrate buffer systems under the usual conditions of amino acid analysis (unpublished observations of Heacock, Williams, Frank, and Adams). It would, however, be amenable to quantitative assay in the older chromatographic system of Hits et al. (14) which employed HCI elution.

While it is possible that further study of the reaction of P5C with acid ninhydrin could yield a chemical assay more suitable than that provided by o-AB, the accuracy and precision of our enzymatic method renders it the one of choice. Earlier, Strecker (15) estimated P5C concentration enzymatically with a preparation from liver of P5C dehydrogenase_ The bacterial enzyme employed by us would seem to offer the advantage of simplicity in preparation and long-term stability. Of course, the specificity of the assay would have to be investigated with care in any applications involving complex milieux such as blood and urine. Studies of human prolinemia have employed o-AB assay for P5C/16), but without authentication of the methodology. In such applications, the validity of enzymatic assay could most easily be studied by parallel reduction of P5C to tritiated proline with sodium borotritide (5) and subsequent quantitation of the radioactive proline.

ACKNOWLEDGMENT

We are grateful to Dr. Paul Canner for statistical advice and analysis of the data, as summarized in the text.

A I -pYRROLINE- 5 -CARBOXYLIC ACID 97

REFERENCES

1. ADAMS, E. (1970) in International Review of Connec t ive T i s sue Research (Hall, D. A., ed.), Vol. 5, pp. 1-91, Academic Press, New York.

2. VOGEL, H. J., AND DAVIS, B. D. (1952) J. Amer. Chem. Soc. 74, 109-112. 3. STRECKER, H. J. 11960)J. Biol. Chem. 235, 2045-2050_ 4. GOOD, N., AND MITCHELL, H. K. 11952) J. Amer. Chem. Soc. 74, 4952-4953. 5. FRANK, L. 11966)Anal. Biochem. 17, 423-433. 6. VAN SLYKE, D. D_, HILLER, A., AND MACFADVEN, D. A. (1941). J. Biol. Chem. 141,

681-705. 7. HILLER, A., PLAZIN, J., AND VAN SLYKE, D. D. (1948). J. Biol. Chem. 176,

1401-1420. 8. PIEZ, K. A., IRREVERRE, F., AND WOLFF, H_ L. 11956)J. Biol Chem. 223, 687-697. 9. FRANK, L., AND RANHAND, B. (1964)Arch. Biochem. Biophvs. 107, 325-331.

10. FRANK, L., AND HOPKINS, 1. (1969) J. Bacteriol. 100, 329-336. 11. STRECKEIa, H. J, (1965) J. Biol. Chem. 240, 1225-1230. 12. LARSON, R. L.,AND BROQUlST, H. P. 11963)Anal Biochem. 5 , 2 5 5 - 2 5 9 . 13. HORECKER, B. L., AND KORNBERG, A. 11948)J. Biol. Clnem. 175, 385-390, 14. HIRs, C. H. W., MOORE, S., AND STEIN, W. H. 11954) J. Amer . Chem. Soc. 76,

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improved chemical synthesis and enzymatic assay ofΔ1-pyrroline-5-car☐ylic acid

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