ANALYTICAL BIOCHEMISTRY 39, 5964 (191)

Quantitative Assay for Hydroxylysine in Protein Hydrolyzatesl

NELLY BLUMENKRANTZ? AND DARWIN J. PROCKOP

Departmetlts of Medicine and Biochemistry, I.~niversity of Pennsylvania, and Philadelphia General Hospital, Philadelphia, Pennsylvania 19104

Received June 17, 1970

Collagen and related glycoproteins are the only animal proteins which contain significant amount’s of the hydroxyamino acids hydroxyproline and hydroxylysine. A number of chemical procedures for the quantitative assay for hydroxyproline have been developed, and these assay pro- cedures have been used extensively on the studies of biosynthesis and metabolism of collagen (see reference 1 for review). Similar studies on the biosynthesis of hydroxylysine have in part been hampered by the lack of rapid and specific assays for this amino acid.

In the present report we present an assay for hydroxylysine which is based on periodate oxidation. Periodate will oxidize a number of amino acids such as scrine, threonine, proline, and hydroxyproline, and periodate oxidation of both serine and hydroxylysine produces ammonia and form- aldehyde (2-4). Part of the specificit#y of the present assay was based on the fact that periodate oxidation of hydroxylysine also produces glutamic semialclehyde and Al-pyrroline-5-carboxylic acid which on further oxida- tion will form a color with p-dimethylaminobenzaldehyde (Fig. 1). Initial experiments indicated t’hat proline gave rise to a similar chromo- phorc in the assay and therefore a preliminary separation of samples by thin-layer chromatography was introduced into the procedure.

MATERIALS

Ground-glass t&es with stoppers or culture tubes, screw-capped with Teflon lillers (Kimaz) , 200 X 25 mm. Tubes were marked with a diamond knife in order to calibrat’e them to a volume of about 8 ml.

Citrate-phosphate buffer. The buffer was prepared by mixing 154 ml

‘This work was supported in part by NIH research grants DE-2623. HD-183, and FR-107 from the U. S. Public Health Service.

f Requests for reprints should be addressed to Dr. Nelly Blumenkrantz, Phila- delphia General Hospital, 34th Street and Curie Avenue, Philadelphia, Pa. 19104.

59

60

$"2N"2 CHOH

i”,

kH2

~HNH

dOOH

BLUMENKRANTZ AND PROCKOP

YHO

j$ CH20 + NH3 + + 4

itO) No104 CHROMOPHORE

FIG. 1. Probable reactions for oxidation of hydroxylysine by periodate. The first reaction product is glutamic semialdehyde, which is probably in equilibrium with Al-pyrrolined-carboxylic acid. As indicated in text, A’-pyrroline-5-carboxylic acid did not give a color with Ehrlich reagent unless it was further oxidized to an unidentified product by periodate.

of 0.15 M citric acid (Fisher Scientific Co.) with 346 ml 0.6 M dibasic sodium phosphate (J. T. Baker Chemical Co.). The pH of the final solu- tion was 7.0.

Standard hydrosylysine solution. S-Hydroxylysine-HCl (mixture of DL and ~~~110, Sigma Chemical Corp.) was prepared at a concentration of 15 ,pg/ml and 15 mg/ml.

Periodate solution. A 0.3 M solution of sodium metaperiodate (Fisher Scientific Co.) in water was prepared, and it was stored for up to 2 months in the dark in a brown bottle covered with aluminum foil.

Extraction solution. The extraction solution was prepared by mixing 250 ml of toluene with 250 ml of isobutanol and 100 ml of n-propanol.

p-Dimethylaminobenzaldehyde or Ehrlich reagent (analytical grade, Coleman, Matheson & Bell Co.). Fifteen ml of isohutanol was added to 4 gm of p-dimethylaminobenzaldehyde in a beaker, and then 4.5 ml of per- chloric acid was added. The solution was stored for several weeks at 4°C in a bottle covered with aluminum foil.

Thin-layer chromatography. Precoated silica gel plates (without fluorescent indicator, 20 x 20 cm with a layer of 250 p, Brinkmann) were used. The solvent system was propanol/water in the proportion 7~3 (v/v).

Sample recovery tube. Chromaflex sample recovery tube (Kontes Glass Co., K-416400) was used for the recovery of samples directly from a developed TLC plate by suction.

ASSAY PROCEDURE

Protein samples were hydrolyzed overnight in 6 N HCl in sealed tubes at 118”C, and the hydrolyzates were evaporated to dryness in a rotatory evaporator.

ASSAY FOR HYDROXYLYSINE 61

Thin-Layer Chromatography. The hydrolyzate was dissolved in 1 ml of 0.02 N NaOH and from 10 to 40 ~1 of the sample was applied to a thin- layer plate. The spot was dried and the system was developed with propanol/water for about 3 hr when glass jars were used, and 2.5 hr with t,he sandwich technique. Standards containing 15 pg of hydroxy- lysine and 30 ,ug of proline in 20 ~1 of 0.02 N NaOH were run in each plate. The section of the plate containing one of the standards was sprayed with 0.3% ninhydrin in butyl alcohol containing 3% glacial acetic acid in order to locate the chromatographic position of the hydroxylysine (Rf of 0.03) and to ensure that good separation from proline (Rj- of 0.30) was obtained. The Chromaflex sample recovery tube was used to remove the area of t’he gel containing the hydroxylysine in the sample and in the second standard. The sample was recovered from the aspirated gel by passing 8 ml of buffer through the recovery tube into a ground-glass tube of the size indicated. Control experiments indicated that, essentially the same results were obtained in the assay when the sample recovery tube was used, and when the gel was carefully removed with a spatula and the hydroxylysine on the gel was extracted directly for assay. However, the sample recovery tube facilitated removal of the gel and it helped prevent losses of small amounts of gel during the transfer.

Hydrosylysine Assay. The volume of the sample was adjusted by add- ing buffer to the mark on the tube to give a final volume of about 8 ml. The reaction was carried out by adding to the tube 0.3 ml of the 0.3 M sodium metaperiodate and the solution was stirred. Then 3.0 ml of the extraction solution (organic phase) was added and the tube stirred on a Vortex mixer. The tube was placed in a test tube rack; the rack was cov- ered with aluminum foil and shaken on a Equipoise horizontal shaking machine for 20 min. The tube was centrifuged for 10 min at low speed in order to separate the aqueous and organic phases. Exactly 2 ml of the organic phase was placed in a test tube, and 0.5 ml of reagent solution containing p-dimethylaminobenzaldehyde was added. The tubes were immediately stirred vigorously. The color was allowed to develop for 15 min at room temperature and the absorbance was read at 565 nm on a Beckman model B spectrophotometer.

RESULTS AND DISCUSSION

The color obtained from hydroxylysine in the assay had an absorbance at 565 nm (Fig. 2). Al-Pyrroline-5-carboxylic acid, which is probably an initial product of the reaction (Fig. l), did not itself give a color with the Ehrlich reagent, but it gave a color with the same absorbance spec- trum as that obtained from hydroxylysine after it was oxidized with

62 BLUMEiYKRANTZ AND PROCKOP

01 400 500 600 w

FIG. 2. Absorbance spectrum of color obtained in assay for hydroxylysine: (upper broken line) assay of 15 pg of authentic hydroxyIysine; (upper solid line) assay of 15 lg of A’-pyrro!ine-5-carhoxylic acid; (lower broken line) assay of 30 ,~g of proline in the procedure not involving TLC step; (lower solid line) absorbance of blank,

periodate. Proline, which is probably oxidized to 81-pyrroline-5-car- boxylic acid by periodate, also gave a color with the same absorbance spectrum as the color obtained from hydroxylysine. Hydroxyproline did

FIG. 3. Assay of hydroxylysine (0) and of A’-pyrroline-5-carboxylic acid (0)

ASSAY FOR HYDROXYLYSINE 63

not give any interference on the assay. Hydroxyproline gave small amounts of color with a maximal absorbance at 565 nm only when the oxidation was carried out in an acid medium with a pH of 3.0 or less.

Assay of pure standards of hydroxylysine indicated that the absorbance obtained was proportional to the amount of hydroxylysine added over a broad range, and an absorbance of 0.050 was obtained with 2 pg hydroxy- lysine (Fig. 3). Under the conditions employed, the color reached maxi- mal intensity 15 min after the addition of p-dimethylaminobenzaldehyde and remained constant for 24 hr.

Further studies indicated that the assay was specific for hydroxylysine when the TLC step was used. The hydroxyamino acids hydroxyglutamic acid, serine, and threonine did not interfere with the assay (Table 1). Also, no significant interference was observed with as much as 1 mg of albumin or 10 mg of trypsin.

TABLE 1 Recoveries of Hydroxylysine in Presence of Amino Acids with

Procedure Present’ed in This Paper

Samplea AbsorbanceG66

Recovery of 15 fig hydroxylysine added to

sample as internal standard (ye)

Hydroxylysine, 15 pg Lysine, 1000 pg Proline, 400 pg Hydroxyproline, 400 pg Serine, 600 pg Threonine, 600 pg Glucosamine, 500 fig Hydrolysate of

albumin, 1000 fig Hydrolysate of

trypsin, 10,000 pg

0.386 0.000 0.000 0.000 0.000 0.000 0.000 0.000

0.000 96.4

96.9 99.4 99.8 98.6

101.2 97.6 98.2

a Values indicate pg of sample placed on TLC plates.

Assay of hydrolyzed rat skin collagen gave a value for hydroxylysine equivalent to 5.5 residues of hydroxylysine per 1000 residues of amino acids. This result agrees with the results of Gross and Piez (5)) indicating a content of 5.7 residues per 1000. Assay of 0.68 mg of cuticle collagen from Ascaris lunabricoides gave no significant amount of color, a result consistent with the fact that no hydroxylysine is found in this particular collagen (6) .

64 BLUMENKRANTZ AND PROCKOP

SUMMARY

A quantitative assay for hydroxylysine in protein hydrolyzates was developed on the basis of periodate oxidation of the hydroxyamino acid to glutamic semialdehyde and Al-pyrroline-5-carboxylic acid, and sub- sequent oxidation of these compounds to an unidentified product which formed a color with p-dimethylaminobenzaldehyde. Since proline gave the same color, a preliminary separation of hydroxylysine from proline was carried out by thin-layer chromatography. The assay will detect 2 pg of hydroxylysine.

ACKNOWLEDGMENTS

The sample of A’-pyrroline5-carborylic acid was a gift from Dr. Harold J. Strecker, Department of Biochemistry, Albert Einstein College of Medicine.

REFERENCES

1. PROCKOP, D. J., AND KIVIRIKECO, K. I., in “Treatise on Collagen” (G. N. Ramachandran and B. S. Gould, eds.). Academic Press, New York, 1968.

2. NICOLET, B. H., AND SHINN, L. A., J. Am. Chem. Sot. 61, 1614 (1939). 3. VAN SLYICE, D. D., HILLER, A., MACFAUYEN, D. A., HASTINGS, A. B., AND

KLEMPEREI, F. W., J. Biol. Chem. 133, 287 (1940). 4. BRAGG, P. D., AND HOUGH. L., J. Chem. Sot. 1958, 4050. 5. GROSS, J., AND PIEZ, K. A., in ‘Calcification in Biological Systems” (R. F.

Sognnacs, ed.) . American Association for the Advancement of Science, Washing- ton, D. C., 1960.

6. WATSON, M. R., AND SILVESTER. N. R., Biochem. J. 71, 578 (1959).