Tyrosine Microcrystals Produced by Digestion of Proteins with

Article pubs.acs.org/crystal

Tyrosine Microcrystals Produced by Digestion of Proteins with Pancreatic Enzymes Alexander McPherson,* Steven B. Larson, and Yurii G. Kuznetsov Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, United States ABSTRACT: If serum albumin from several animals, including humans, at concentrations greater than about 60 mg/mL is combined with an aqueous extract of porcine or human pancreas, then massive amounts of microcrystals develop. Both raw blood serum, as well as a range of concentrated, pure proteins, when combined with the extract produced identical crystals. Morphologies of crystals and their aggregates did, however, show some dependence on the starting protein material. Crystals could be dissolved by boiling in water and recrystallized. By X-ray diffraction we showed the crystals to be Ltyrosine crystals, presumably a degradation product of the starting proteins. Amino acid analysis, supported by mass spectrometry, of even well washed crystals, however, consistently showed them to be composed of 20% to 30% of other amino acids. An atomic force microscopy investigation of the crystals revealed that the crystal surfaces were persistently coated with spherical particles about 3 nm in diameter. The particles are apparently micellar arrangements of oligopeptides. Some implications of our results with regard the appearance of tyrosine crystals in human tissues under pathological conditions, in tumors, and in natural products such as foods are discussed.

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Given that the “ancient trypsin” solution that successfully yielded the crystals was originally derived from porcine pancreas, we ultimately had the wit to expose a 200 mg/mL solution of BSA to an aqueous extract of porcine pancreatin, the acetone powder of the pancreas. The results were quite remarkable. Not only was the identical microcrystalline product obtained as previously, but in higher yield and in less than 24 hours time. If the pancreatin extract was concentrated 5-fold, then the time of crystallization was reduced to only 3 or 4 h. Blood sera from several animals were also exposed to the pancreatin extract. Masses of the same microcrystals (Figures 1c and d) were obtained. Finally, we found that many other pure proteins when treated with the pancreatin extract also yielded the crystals. Questions that the appearance of the microcrystals posed were their (a) composition, (b) physical properties, (c) structure, and (d) potential significance.

INTRODUCTION In the course of exploring the use of oligopeptides to promote or enhance the crystallization of macromolecules,1,2 we created mixtures of peptides by proteolytically digesting common proteins. Among the proteins were bovine serum albumin (BSA) and human serum albumin (HSA). In these experiments, entirely by chance, we observed that HSA or BSA at high concentrations (>100 mg/mL), when exhaustively digested for two weeks at 45 °C with a 15 year old commercial porcine trypsin solution generally used in cell culture work (Gibco catalog no. 15090−046, lot no. 1133099, expiration date 10/2003) yielded a copious microcrystalline product. Examples of the crystals are seen in Figures 1a and b. Though a microscope is necessary to visualize the many individual blade-like crystals that make up the clusters, when the experiment is carried out in a test tube, the masses of crystals are so thick that they can readily be seen with the naked eye. We were subsequently unable to reproduce this result with the same (according to the manufacturer) trypsin solutions currently on the market, nor with pure trypsin, which produced a stiff gel, nor with chymotrypsin, nor with mixtures of pure trypsin and chymotrypsin, which yielded precipitates. The “ancient trypsin” preparation that initially produced the crystals did exhibit on SDS-PAGE protein bands in addition to those corresponding to pure trypsin. Some of the additional bands were of higher molecular weight and could not, therefore, have been degradation products of trypsin. This was the first clue that multiple enzymatic factors might be involved in the production of the crystals. © 2012 American Chemical Society

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EXPERIMENTAL PROCEDURES Pancreatic Extract. Pancreatic extract was made by stirring 1 g of porcine pancreatin (Sigma Biochemical Co., St. Louis, MO; catalog no. P-1750, grade VI, lot no. 58F-0575) with 10 mL of distilled water for 30 min at room temperature and centrifuging the mixture at 8K in an SS-34 rotor for 20 min to remove insoluble material.

Received: March 29, 2012 Revised: May 31, 2012 Published: June 5, 2012 3594

dx.doi.org/10.1021/cg300420e | Cryst. Growth Des. 2012, 12, 3594−3602

Crystal Growth & Design

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masses and manipulated because of their size) were drawn into 0.7 mm diameter quartz capillaries and sealed at both ends with bee’s wax to maintain hydration.3 X-ray diffraction images were recorded using a Rigaku RU-200 generator producing CuKα radiation and fitted with Osmic mirrors. The detector was a Rigaku CCD detector. The exposure times varied from 2 to 5 min and the images were collected at room temperature. Atomic Force Microscopy (AFM) Analysis. Samples of microcrystals suspended in H2O were applied to substrates of freshly cleaved mica, fixed with 5% glutaraldehyde for 5 min, and scanned in liquid in a 75 μL fluid cell, or dried in a stream of nitrogen gas and scanned in air. The procedures were basically the same as described previously for the AFM analyses of protein and virus crystals4,5 and for individual viruses and cells.6,7 The instrument we used was a Nanoscope III AFM (Veeco Instruments Inc., Santa Barbara, CA). Images were collected in tapping mode using silicon nitride tips in liquid and silicon tips in air. In the images presented here, dark areas or objects represent very small heights above the background, while light areas correspond to features high above the substrate surface. Characterization of the Composition of the Crystals. Staining. Coomassie brilliant blue dye was added to a suspension of the crystals in water until the solution was only a pale blue. After 30 min, the sample was examined using a 40× magnification microscope to determine the color of the crystals with respect to the surrounding fluid. Solubility. A mass of microcrystals collected by centrifugation was suspended in 1 mL of water and stirred for 12 h at room temperature. The sample was then centrifuged in a microfuge for 10 min to sediment any remaining crystals. The optical density of the supernatant at 280 nm was then measured in a Pharmacia spectrophometer (Ultraspec 2100). The peptide concentration in the supernatant was also determined by the method of Lowry8 using a kit supplied by Bio-Rad Corp., Hercules, CA. Composition. The amino acid composition of the microcrystals, after washing with water and collection by centrifugation, was determined after 12 h of hydrolysis in HCl at 100 °C by Alphalyse Co. (Palo Alto, CA). Crystals were dissolved in water to their maximum solubility, and also in formamide. Solutions were analized by HPLC on a C18 column, followed by mass spectrometry at the UC Irvine Chemistry Department facility for mass spectrometry using MALDI-TOF technology. Gel Analysis. Microcrystals were readily dissolved in 1% sodium dodecyl sulfate (SDS) and run on SDS polyacrylamide gels of 10% and 15% polyacrylamide composition as described by Laemmli.9 Precast gels were purchased from Bio-Rad, Hercules, CA. Gels were stained with Coomassie brilliant blue and destained in acetic acid−water. Human Pancreatic Extract. A portion (43.3 g wet weight) of human pancreas obtained from autopsy was mascerated in a chilled Waring blendor with 100 mL. of cold phosphate buffered saline (PBS) and centrifuged in an SS-34 rotor at 8000 rpm for 12 min. The supernatant was set aside on ice. The insoluble material was extracted in the blendor a second time with 30 mL of PBS, centrifuged as before, and the supernatant added to the first. The combined supernatants, having a total volume of about 100 mL were centrifuged at 10 000 rpm in an SS-34 rotor for 30 min to further remove insoluble material. The supernatant was the pancreatic extract used in crystallization experiments.

Figure 1. (a and b) Light microscope photographs (200× magnification) of bundles and sheaves of fine needle crystals obtained from bovine serum albumin exposed to an extract of porcine pancreatin. The crystals exhibit strong birefringence. (c) Microcrystals that appear the same as those shown in panels a and b but produced by exposure of fetal calf serum to pancreatic extract. (d) Crystals produced from raw horse serum. (e and f) Examples of the recrystallization of the material derived from bovine serum albumin seen in panels a and b by vapor diffusion. Images in panels c and d were obtained using nonpolarized light, others using polarized light.

Crystallization and Recrystallization. For standard assays for crystal formation, crystals were grown by mixing 4 parts of 200 mg/mL bovine serum albumin (BSA) fraction V, heat shock treated (FisherBiotech, Atlanta, Ga., catalog no. BP 1600, lot no. 980036) in water with 1 part of the pancreatin extract and incubating the mixture at 37° to 45 °C for 24 to 48 h. Crystals continue to accumulate for several days in this temperature range. In nonstandard assays with other proteases, protease sources, other proteins, or serums the ratios were the same but crystalliztion times were from a few hours to ten days or more. Recrystallization was carried out by vapor diffusion3 in plastic Cryschem plates (Hampton Research Co., Aliso Viejo, CA) with microdrop volumes of 6 μL. The drops were composed of equal volumes of a saturated solution and a reservoir solution provided by kits from Hampton Research, specifically Crystal Screen I and Index. Crystallization trials were carried out at room temperature and evaluated with a Bausch and Lomb 40× magnification disecting microscope with a zoom lens, and a 200× magnification Olympus BH microscope with cross polarizers. Photographic images were recorded using an Olympus OM-2 camera mounted on the Olympus microscope. The saturated solutions used in the recrystallization experiments were prepared in two ways. In the first case a small amount of acetic acid was added to masses of microcrystals suspended in water in a glass test tube. The test tube was then held under the hot water tap until dissolution of the crystals appeared complete. The solution was then centrifuged at room temperature to remove any remaining microcrystals. In the second case, masses of microcrystals in a glass test tube were submerged in vigorously boiling water for about 10−15 min which also affected dissolution. The latter solution was then centrifuged well before being used for recrystallization. X-ray Diffraction Analysis. Bundles and masses of microcrystals (single crystals could not be isolated from the 3595



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Microcrystals from Blood Sera. To determine whether microcrystals could be obtained directly from animal blood serum, pancreatin extract was combined in a 1:4 ratio with serum from humans (Sigma Biochemicals, St. Louis, MO), adult cattle (Sigma Biochemicals, St. Louis, MO), fetal calf serum (Invitrogen, Grand Island, NY), horse serum (EquitechBio, Inc. Kerrville, TX), and sheep serum (Equitech-Bio, Inc. Kerrville, TX).

To test the human pancreatic extract for its capacity to produce microcrystals from albumins, the extract was combined in a 1:4 ratio with 200 mg/mL BSA in H2O and with 100 mg/ mL HSA in H2O and incubated at 37 °C. The samples were 1 mL in final volume. Testing of Other Proteases for the Capacity to Produce Microcrystals. BSA was exposed to a variety of different proteases in addition to trypsin and the porcine pancreatic extract. The proteases were chymotrypsin, carboxypeptidase B, subtilisin, papain, pepsin, proteases from aspergillus sojae and Aspergillus niger and proteinase K. All proteases used in the experiments described here were from Sigma Biochemicals except carboxypeptidases A and B which were purchased from CHEM-IMPEX, Wood Dale, IL and Calzyme, San Louis Obispo, CA respectively. The proteases were generally dissolved in water to concentrations of 25 mg/ mL where possible, or their maximum concentrations otherwise. The two carboxypeptidases were not soluble in water and were dissolved in 0.3 M NaCl. To test for a protease’s crystallogenic capacity, the protease solution was combined with 200 mg/mL BSA in water in a 1:4 ratio and incubated at 37 °C. Samples were examined by microscope over a 30 day period. Mixtures of Proteases. In reconstituting the active proteolytic principles of the porcine pancreatic extract four different proteases were combined in all possible double and triple combinations. The four proteases were porcine trypsin, bovine chymotrypsin, and bovine carboxypeptidases A and B. The trypsin and chymotrypsin stock solutions were 25 mg/mL in protease while the two stock carboxypeptidase solutions were composed at 5 mg/mL protease. Mixtures were made by combining equal amounts of the component proteases. Assays for the production of microcrystals by the protease mixtures were carried out in 3 mL glass tubes containing 0.5 mL of 200 mg/mL BSA in water and 0.125 ul of the protease mixture. Protease Inhibition. To narrow the identity of the crystallogenic components in the pancreatic extract, crystallization assays were carried out using a variety of known protease inhibitors. For an assay, porcine pancreatic extract was preincubated with an inhibitor for 15 min at room temperature. The protease−inhibitor mixture was then added to a 200 mg/ mL BSA in water solution in the ratio of 1:4. Concentrations of the inhibitors were at least several times that necessary to inhibit an equal amount of a pure protease at 25 mg/mL concentration. Crystals of Proteins Other Than Albumins. Proteins having no significant amino acid sequence identity were dissolved in H2O or PBS (microcrystals will readily form in both water and PBS) to the maximum concentration that could be obtained at room temperature and centrifuged to remove insoluble material. With the exception of the albumins that were readily soluble in excess of 800 mg/mL, the remaining proteins had solubilities generally below 100 mg/mL, and some were soluble to no more than a few mg/mL. The clarified protein solutions were then concentrated using Amicon CS15 minicon concentrators (Millipore Corp., Bedford, MA). Some proteins produced precipitate upon concentration and this was removed by centrifugation. In a practical sense, all proteins were brought to the highest concentration that we were able to obtain. To test whether the final concentrated protein could produce microcrystals when combined with pancreatic extract, the concentrated protein solutions were individually combined with pancreatic extract in a 4:1 ratio.

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RESULTS Crystallization. The majority of the work reported here was carried out using crystals prepared from serum albumin (reviewed by Peters, 1996), because of their ready availability and high solubility. The capacity to form microcrystals upon proteolytic degradation, as we show below, is not a specific feature of the albumins, however, but is an inherent property of many, but not all proteins when highly concentrated. Concentration of the pancreatin extract accelerated the appearance of crystals, as did seeding with previously grown crystals. Seeding, however, was not essential. When crystallization was complete, which required several days, the test tubes containing the albumin/pancreatin extract mixture were heavily congested with masses of microcrystals. Crystals were stable for at least two years at room temperature. Crystallization appeared unaffected by the inclusion of reducing agents such as mercaptoethanol, or divalent metal ions such as Ca2+ or Mg2+. Minor differences in detailed morphology were present such as the sizes of the crystal clusters and the lengths of the individual crystals making up the clusters, depending on the source of protein. It was significant that when blood sera of several animals were mixed with porcine pancreatic extract the same microcrystals were obtained as from the purified albumins (Figure 1c and d). The concentration of albumin in animal serum is only about 40 mg/mL,10 which is considerably lower than the 200 mg/mL albumin solutions that we had used up to this point. The total protein concentration in serum is, however, much higher than 40 mg/mL. This suggested that degradation products of proteins other than albumins might contribute to the growth of crystals. Once crystals from any source were dissolved to produce a concentrated solution they could then be recrystallized by vapor equilibration11 against any number of conditions that used as precipitants both concentrated salts, MPD, and PEG. Recrystallization produced bundles of longer, and somewhat larger crystals, but otherwise of similar cross section as the original crystals (Figure 1e and f). Crystallization with salt solutions was frequently preceded by phase separation (oiling out) in the mother liquors. Chemical Composition. The only materials present in the initial reaction mixture, and subsequently in the crystallization mother liquor were pure albumin dissolved in water and the water extract of porcine pancreatin. There were no buffers, salts or other chemicals included. The yield of crystals was such that they could not be explained by some component of the pancreatin extract. In addition, crystals had been produced with the “ancient trypsin” from Gibco, and (see below) were later produced using a mixture of pure enzymes rather than the pancreatin extract, further demonstrating that the crystals did not originate from pancreas components. The crystals, therefore, had to be derived from albumin. Intact albumin yields no crystals except in rare conditions,12,13 and those crystals are not like the microcrystals observed here. Because 3596



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the pancreas extract is rich in proteases14 the only reasonable explanation for the microcrystals was that they were composed of some proteolytic product of the albumin. The microcrystalline needles, though very thin (0.25−0.50 μm in thickness, but sometimes as much as a half millimeter in length) are strongly birefringent (Figure 1a and b) indicating that their internal structure is highly anisotropic, consistent with their habits. They invariably grow in dense clusters. The crystals stained dark blue with Coomassie blue dye. They are marginally soluble in water with an equilibrium concentration at room temperature equivalent to protein, as measured by Lowry’s procedure8 of about 2−3 mg/mL, and an optical density at 280 nm of about 2.5. The crystals when run on SDSPAGE exhibited no bands behind the bromphenol blue dye front, showing there to be little or no large degradation products present in the crystals. Amino acid analyses of crystals produced from bovine and human albumin consistently showed that the crystals were composed of at least 70% tyrosine, but also numerous other amino acids to be present in total amounts as high as 30% of the sample. HPLC followed by mass spectrometry indicated oligopeptides to be present but with none predominant. X-ray Diffraction Characterization of the Crystals. Representative diffraction photographs from capillary mounted masses of crystals and crystal sheaves are shown Figure 2. As

Table 1. Bragg Spacings Giving Rise to the Diffraction Rings in Figure 3 Bragg spacing (Å)

intensity

5.73 5.31 4.96 4.50 4.34 4.10 3.61 3.47 3.42 3.29 3.12 2.91

strong weak strong medium very strong medium strong strong strong medium weak weak

sistency remained, however, that amino acid analysis, HPLC, and mass spectrometry consistently indicated the presence of substantial amounts of other amino acids and oligopeptides. Atomic Force Microscopy Characterization of Crystals. Crystals were subjected to analysis by AFM with the objective of visualizing the surface structure of the crystals.4,5 The mother liquor surrounding the crystals, that liquid from which the crystals were grown, was similarly examined to determine what entities might contribute to the crystals’ growth.11 Typical AFM images of the crystals are shown in Figure 3. Even at high magnification, sharp edges and flat faces characteristic of true crystals were present. The crystals exhibited a small, but flat face perpendicular to the long direction at one end (Figures 3a - c) and a pointed tip at the other (Figure 3f) indicating that growth proceeded principally or entirely in one direction as well as the probability of a polar axis in the long direction. Seen in Figures 3 d and e are growth steps and macrosteps. The most outstanding features were stripes running parallel to the crystals’ long axes (Figure 4). The stripes are closely packed laterally. The widths or diameters of the stripes are about 3 nm on crystals dried in air and about 4 to 5 nm in fluid. There was, however, no clearly observable crystallographic period along the principal crystal axis. The background surrounding the crystals and the crystal surfaces were informative. The substrate, in those cases where the crystals were in equilibrium with their initial mother liquor, was covered with spherical entities of more or less uniform size, having diameters of about 3 nm. A striking feature of the microcrystals was the affinity of the spherical particles for the crystal surfaces. As can be seen in Figure 5a−e, the surfaces were virtually coated with the particles. The particles adhered to the surfaces even when the crystals were repeatedly washed with water or PBS. Our assumption is that the spherical particles, consistent with biochemical and other physical data, are composed of relatively short oligopeptide products of proteolysis. Indeed, other instances of proteolytic fragments coalescing into spherical “micellar” particles have been reported.15 What we find most remarkable here is the degree and extent of association with the crystal surfaces. Also intriguing is that occasionally, as seen for example in Figure 5d and e, the oligopeptide particles, which are of rather uniform diameter, arrange themselves in linear and occasionally two-dimensional arrays. Examination of AFM images suggests that the particles align themselves and selfassociate along step edges, or growth steps on the crystal

Figure 2. (a) Still X-ray diffraction photograph made from a mass of wet crystals. The microcrystals have random orientations thereby effectively yielding a powder pattern. (b) A 0.50 degree oscillation photograph of a single sheaf, or bundle of the crystals that has some degree of crystal orientation.

expected, the packed crystals yielded patterns corresponding to powder diffraction patterns (Figure 2a), and photographs from sheaves (Figure 2b) similar, but with some additional detail. From the Bragg angles for the rings, one could, in principle, obtain estimates of the unit cell parameters of the crystals. Table 1 indicates, for each ring observed in the diffraction patterns, the responsible Bragg spacing in the sample. A search of the literature eventually led us to a report of (Ramachandran and Natarajan 2002) titled “Growth of some urinary stone constituents: I. In-vitro crystallization of L-tyrosine and its characterization”.42 Comparison of the X-ray pattern characterized quantitatively in Table 1 with their powder diffraction pattern recorded from microcrystals of L-tyrosine grown in gels, in vitro, showed the two intensity patterns to be virtually identical over the entire resolution range reported. The correspondence unequivocally demonstrated that the crystals we produced through degradation of pure proteins, raw serum, and protein mixtures were L-tyrosine crystals. The incon3597



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Figure 3. AFM images of crystals derived from BSA showing the flat surfaces, straight edges, and sharply defined angles characteristic of true crystals. Also seen in d−f are growth steps and macrosteps as are observed on conventional crystals. The scan areas for the images are (a) 3 μm × 3 μm, (b) 1 μm × 1 μm, (c) 500 nm × 500 nm, (d) 500 nm × 500 nm, (e) 500 nm × 500 nm, and (f) 1 μm × 1 μm.

Figure 4. At this magnification, the only features indicative of order on the crystal surfaces were stripes, or bands parallel with the long dimension of the crystals. These had relatively consistent widths of about 3 nm on air-dried samples. There were no clear indications of periodicity along the long axis of the crystals. The scan areas for the images are (a) 1 μm × 1 μm, (b) 500 nm × 500 nm, and (c) 500 nm × 500 nm.

Figure 5. (a−e) Surfaces of washed tyrosine crystals can be seen coated with spherical particles composed of oligopeptide degradation products. In (b), (d) and (e) the particles align in linear and occasionally two-dimensional arrays on the crystal surfaces, apparently ordered by underlying growth steps and macrosteps of the tyrosine crystals. In (f) is a thoroughly washed crystal on which the tyrosine crystal lattice is just visible. Scan areas are (a) 500 nm × 500 nm, (b) 250 nm × 250 nm, (c) 250 nm × 250 nm, (d) 500 nm × 500 nm, (e) 500 nm × 500 nm, and (f) 1 μm × 1 μm.

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amino peptidases, and sulfhydril proteases had no visible effect on the reaction. Significantly, EDTA and 1,10 phenanthroline, both known inhibitors of metaloproteases, shut down crystal formation completely. The predominant metaloproteases of the pancreas are carboxypeptidases A and B, both of which are zincdependent enzymes. Reconstitution. In an effort to reconstitute the activity present in the pancreatic extract, all combinations of trypsin, chymotrypsin, carboxypeptidase A, and carboxypeptidase B in pairs and multiples were formulated in buffer. Using BSA as the protein substrate only one combination reproducibly produced the tyrosine microcrystals, and that was when all four proteases were simultaneously present. Combinations of three proteases or less, lacking the forth, were not effective. Thus it appears that all four proteases are necessary. This is in some disagreement with the inhibition experiments that indicated chymotrypsin to be unimportant (no inhibition of crystals by TPCK), but nonetheless, chymotrypsin appears to be a necessary component in the reconstituted enzyme mixture. The combination of all four enzymes, we observed, produced a smaller yield of crystals than did pancreatic extract, thus it remains possible that some other proteases from the pancreas may be involved that can enhance crystal formation, but that are not essential. In passing, and in retrospect, it appears probable that the carboxypeptidases were the contaminants in the “ancient trypsin” from Gibco that initiated this investigation, contaminants that have apparently been eliminated from the preparations currently on the market. The finding that trypsin and carboxypeptidases were necessary components of the pancreatic extract responsible for creation of the constituents of the crystals is explicable in terms of enzyme specificity.17,18 Trypsin and carboxypeptidases B and A operate in tandem. Trypsin cleaves polypeptides to produce fragments having carboxy terminal lysine and arginine residues. Carboxypeptidase B cleaves the terminal arginine and lysine residues from the peptide termini thereby allowing degradation by carboxypeptidase A to proceed. Chymotrypsin on the other hand produces peptides with aromatic or large hydrophobic residues, including tyrosine, at carboxy termini, and these are then freed by carboxypeptidase A. One might have expected that chymotrypsin plus carboxypeptidase A alone might have been sufficient to produce free tyrosine and therefore the crystals, but this was not the case. We explored the possibility that other pure proteases might be substituted for the pancreatic extract. Indeed we did obtain tyrosine crystals with thermolysin, papain, subtilisin, and several other bacterial and fungal proteases. In all of these experiments, however, the results were sporadic, often irreproducible, required long periods of incubation time, and never produced the yields of crystals that the pancreatic extract did. Protein Substrates. Because of early indications that the formation of microcrystals might not be unique to the albumins, we investigated the digestion of a panel of proteins to see whether crystals were produced. As seen in Table 3, more than 50% of the proteins yielded masses of tyrosine microcrystals. Some are shown in Figure 6. The crystals were again more or less the same in appearance under the microscope, though detailed morphologies of clusters and masses were generally specific to the protein substrate. From Table 3, it is clear that not all the proteins tested yield microcrystals, the hemoglobins in particular, but most do if they are sufficiently concentrated.

surfaces. Because the step edges are at some locations patterned, the coating of particles may assume a similar appearance. The interaction of the particles with the surfaces, undoubtedly affects the growth properties of the crystals, i.e. their growth rates and the detailed morphologies of the crystals. The phenomenon of surface poisoning, as it is commonly called, has been widely investigated for a vast number of crystal systems.16 The attraction of the proteinacious particles and their ordering by the growth steps further suggests that the tyrosine crystals could conceivably serve as heterogeneous nucleants for protein crystallization. If microcrystals obtained by recrystallization from PEG solutions were further washed many times with warm water, then occasionally areas on the crystal surfaces could be cleared of the oligopeptide spheres. Those areas, illustrated by Figure 5f, then revealed the true surfaces of the tyrosine crystals. In some AFM images, it was just possible to resolve the actual tyrosine crystal lattice. Inhibition of Pancreatin Extract Activity. Formation of microcrystals provided a rapid and simple assay for the enzymatic activities essential to producing the crystal constituents. Because trypsin, chymotrypsin, and carboxypeptidases A and B were individually insufficient to produce crystals (see below), the suspicion was that the responsible proteases were either a minor pancreatic protease, or that the activity resided in a combination of the prevalent proteases. To assist in identifying which enzymatic activities might be involved, the reaction of pancreatin extract with albumin was tested in the presence of a panel of protease inhibitors. The results of the experiment, presented in Table 2, indicate that all of the trypsin inhibitors eliminated the formation of Table 2. Inhibition of Pancreatic Enzymes inhibitor soy bean trypsin inhibitor turkey egg trypsin inhibitor p-methyl sulfonyl fluoride p-chloromercury benzoate EDTA TPCK bestatin benzamidine pepstatin 1,10phenanthroline protease inhibitor cocktaila control: no pancreatic extract control: no inhibitor a

enzyme inhibited trypsin

results

trypsin

no crystals grew until 5 days when some began to appear no crystals grew

serine proteases

no crystals grew

cysteine proteases

crystals grew

metaloproteases chymotrypsin amino peptidases serine proteases acid proteases metaloproteases

no crystals grew crystals grew crystals grew crystals grew crystals grew no crystals grew

most proteases

no crystals grew no crystals grew crystals grew

Sigma Biochemical Co., catalog no. P8849.

microcrystals for at least 5 days and in most cases entirely. We knew, however, that pure trypsin did not by itself yield the crystals when mixed with concentrated albumin. This observation seemed to eliminate the likelihood that a single minor protease was responsible for the crystals, as trypsin was clearly implicated. Inhibitors of chymotrypsin, acid proteases, 3599



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them most rapidly and in greatest amounts was insulin, a protein also produced in the pancreas. Microcrystals Using Human Pancreas Extract. As described in the Methods section, we made an extract of human pancreas obtained from autopsy that was in most ways similar to the porcine pancreatin extract used in other experiments. The question we were addressing was whether there existed a combination of the crystallogenic enzymes present in human pancreas corresponding to that in porcine pancreas. Standard assays were carried out to see whether microcrystals could be produced with the human pancreatic extract After 72 h, no crystals were observed by microscopy in either the BSA or HSA assays. After a week’s time (about the same time as was required for the “ancient trypsin”); however, crystals the same in appearance to those produced by porcine pancreatin extract were observed in both the BSA and HSA samples, with the HSA sample congested with the microcrystals. The combination of BSA with the human pancreatin extract produced microcrystals, but also large quantities of spheroids (Figure 6h). The spheroids, however, show the distinctive Maltese cross pattern under polarized light indicative of paracrystalline aggregates and aggregates of crystals too small to be seen. Thus the secretions of the human pancreas have the capacity to produce similar results as we saw using porcine pancreas, though the rates of crystal production were less. This may reflect a lower concentration of the crystallogenic enzymes in human tissue extract with respect to porcine pancreatin. An interesting observation was that if we first made human pancreatin, the acetone powder of human pancreas, and then made the proteolytic extract from the pancreatin (as was done for the porcine material) a noticeable increase in efficiency was obtained.

Table 3. Proteins Other than Albumins that Produced Crystals After Exposure to the Porcine Pancreatin Extract protein

source

results with pancreatin extract

hemoglobin concanavalin A catalase glucose isomerase DNase Lipase α amylase α amylase albumin ovalbumin γ globulins hyaluronidase hemoglobin cytochrome C hemocyanin β lactoglobulin canavalin edestin Bence−Jones protein

human jack bean bovine liver bacteria bovine pancreas fungus bacteria porcine bovine serum hen egg bovine bovine horse horse horseshoe crab bovine milk jack bean hemp seed human

heavy precipitate viscous liquid, light precipitate crystals viscous liquid, light precipitate crystals clear clear, light precipitate clear, light precipitate crystals crystals crystals clear, light precipitate heavy precipitate crystals crystals crystals crystals clear crystals

Some of the proteins could not be highly concentrated, such as the alpha amylases, edestin and Concanavalin A. Inspection of Table 3 suggests some correlation (though not exact, for example, glucose isomerase) between protein concentration and the propensity to form crystals. This is logical since crystallization is a concentration dependent phenomenon. If α amylase, edestin, and concanavalin A were highly concentrated, then they too might yield the crystals. Interestingly, of all the proteins that produced tyrosine crystals, that which yielded

Figure 6. Microcrystals from proteins other than albumins produced by reaction of their concentrated solutions with porcine pancreatic extract. In (a) canavalin, (b) DNase, (c) catalase, (d) hemocyanin, (e) gamma globulin, (f) cytochrome c, (g) beta lactoglobulin, and (h) crystals and spheroids marked by Maltese crosses produced from BSA but using an extract of human pancreatin. Nonpolarized light was used to obtain all images except h. 3600



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DISCUSSION Reports of tyrosine crystals in natural products, such as foods, and in vivo in plant and animal tissues that are identical to those described here have appeared in the literature for over 100 years.19 In virtually all of those reports, the common features were natural protein hydrolysis or proteolysis, or the excessive accumulation of tyrosine. For example, tyrosine crystals are the white material in cheeses (Gouda, Roquefort, Parmesan) that give it its “crunchy” texture.19−21 Crystals of tyrosine are also common in prosciutto and other dried hams and produce the white films often associated with frozen and dried meats of many varieties.22 They have been found in fermentation products such as soy paste.23 Perhaps more important, tyrosine crystals have been observed in animal tissues, including those of humans under pathological circumstances. They are seen in the urine of patients with liver damage,24 and those suffering from one of the three forms of genetic tyrosinemia.25,26 In the latter cases, the crystals may also be found in the corneas of the eyes and in skin patches on the hands and soles of the feet.26−28 They have been found in gastric mucosa29 and vocal cord tissue.30 Tyrosine crystals have also been described in association with numerous kinds of tumors but particularly those of the parotid glands31−33 and the salivary glands.34−37 This is noteworthy because those glands also generate digestive enzymes similar to those produced by the pancreas. Thus the proteolytic process that we employed to obtain the crystals may parallel natural processes that occur in some living tissues. The tyrosine crystals have not, however, been found exclusively in tumors of these glands but have also been reported as occurring with myelogenous leukemia,38 in the bone marrow,39 sweat glands,40 and in fibrous meningioma, a brain tumor.41 The tyrosine crystals that we describe, which appear to be the same as those found in tissues and natural products, are in general not the same as any of the several crystal forms normally obtained by conventional laboratory crystallization techniques. They seem to be particular to in vivo, post mortem, or natural product aging processes (dried meats, cheese). The one exception is the crystallization by Ramachandran and Natarajan42 with whom we compared our X-ray diffraction data. In that case, however, the crystals could only be obtained by an intricate procedure in which the crystals were grown in a gel. We believe that the unique form and properties of these crystals, derived directly from proteins, likely arise from the profound association of the oligopeptide proteolytic products with the crystal surfaces. The interactions between the surfaces and the spherical oligopeptide particles are obviously substantial. They are disrupted by only the most vigorous and extensive washing. Furthermore, we observe that some detailed morphological features of the crystals are modified as a consequence of the protein source, hence by the particular oligopeptide composition of the particles. We would expect similar source dependence for tyrosine crystals appearing in vivo, in tissues. It is noteworthy that many papers in the literature regarding tyrosine crystals in vivo pointedly refer to them as “tyrosinerich crystals.” This is undoubtedly because the crystals, though having a lattice composed of tyrosine alone, are strongly associated with oligopeptides on their surfaces. Thus, it seems evident that in vivo polypeptide degradation products of proteins are implicated. This may be due to enhanced transport of tyrosine to the surfaces by the oligopeptides, or to

maintaining the integrity of the crystals, perhaps by restricting their solubility once they have formed. It is significant perhaps that the optimal production of the crystals was achieved when the starting protein was exposed to an extract of pancreatin. Clearly, the unique combination of enzymes present in the pancreas was more efficient than any single protease from any source, or any reconstituted mixture of proteases that we could compose. Logically then, it might be expected that the tyrosine crystals would appear in tissues where such enzymes are present, for example, the pancreas itself. Although we have not encountered (as yet) reports of tyrosine crystals in pancreatic tissue, we do note again, however, the numerous descriptions of the crystals in tumors of the parotid and salivary glands, which produce proteolytic enzymes similar to those of the pancreas. As noted above, insulin in high concentration, when combined with the pancreas extract, produced masses of tyrosine crystals at a very rapid rate, as high as any pure protein that we tested. Since insulin is also a pancreatic product, we might further have expected reports of tyrosine crystals derived from insulin in pancreatic tissue. Perhaps, in fact, they have been observed, but simply remained unidentified or unreported. Finally, it is worth remarking that when various proteins are exposed to the same amount of pancreatic extract, there is a wide difference in the rates at which the crystals appear, and the eventual quantities. Insulin and β-lactoglobulin from milk yielded massive amounts of crystals in a few hours, while other proteins required many days. Serum albumin and various immunoglobulins yielded copious amounts of crystals, while ovalbumin yielded only a few clusters. There are also some proteins that, even at high concentrations, produced no crystals at all, even though they did contain tyrosine. The hemoglobins were noteworthy in that regard. Presumably the rates and yields are not simply a function of the amount of tyrosine contained in the starting material, but a consequence of either the secondary and tertiary structure of the starting protein, or the composition of the proteolytic fragment particles and how these affect the nucleation of the crystals. Although observations on hemoglobins might suggest that proteins composed principally of α-helix are most refractile, serum albumin is also largely composed of α-helix. Thus the relationship between rate, quantity, and the structures of the starting protein and its fragment particles must be more complicated.

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AUTHOR INFORMATION

Corresponding Author

*Address: Department of Molecular Biology and Biochemistry, University of California Irvine, 560 Steinhaus Hall, Irvine, CA 92697-3900. Tel: (949) 824-1931. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Dr. John Greaves of the UCI Chemistry Department and Courtney Streeter for their assistance with HPLC and mass spectrometry analysis of the crystals and Mr. Sheng-Chieh Chang for aid in preparing figures. Thanks also to Ms. Stephanie Olamendi who was instrumental in obtaining autopsy specimens. This research was supported by a grant to AM from the NIH GM080412. 3601



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(28) Klavins, J. V. Pathology of amino acid excess. VII. Phenylalanine and tyrosine. Arch. Patol. 1967, 84, 238−250. (29) Lee, S. Y.; Choi, Y. S.; M.A., L. Formation of tyrosine crystals in the gastrointestinal tract: uncommon putrefactive artifact. Korean J. Leg. Med. 2000, 24 (1), 20−24. (30) Maurice, Y. M.; Sheard, J. D.; Helliwell, T. R. Tyrosine-rich crystalloids in vocal cord mucosa. J. Clin. Pathol. 2008, 62 (1), 95. (31) Bullock, W. K. Mixed tumor of parotid gland with tyrosine crystals in the matrix. Am. J. Clin. Pathol. 1953, 23, 1238−1239. (32) Carson, H. J.; Rasian, W. F.; Castelli, M. J.; Gattuso, P. Tyrosine crystals in benign parotid gland cysts: Report of two cases diagnosed by fine-needle aspiration biopsy with ultrastructural and histochemical evaluation. Am. J Clin. Pathol. 1994, 102 (5), 699−702. (33) Wright, R. G. Tyrosine crystals in a parotid pleomorphic adenoma in a Vietnamese boat person. J. Clin. Pathol. 1983, 36 (2), 237−238. (34) Friedman, I.; Spilg, W. G.; Russel, T. S. Tyrosine crystals in salivary gland tumors. J. Clin. Pathol. 1982, 35 (1), 120−121. (35) Gilcrease, M. Z.; Nelson, F. S.; Guzman-Paz, M. Arch. Pathol. Lab. Med. 1998, 122, 644−649. (36) Gould, A. R.; Van Arsdall, L. R.; Hinkle, S. J.; Harris, W. R. Tyrosine-rich crystalloids in adenoid cystaic carcinoma: histochemical and ultrastructural observations. J. Oral. Pathol. 1983, 12 (6), 478− 490. (37) Thomas, K.; Hutt, M. S. Tyrosine crystals in salivary gland tumors. J. Clin. Pathol. 1981, 34, 1003−1005. (38) Ayers, W. W.; Starkey, N. M. Studies on Charcot−Leyden crystals. Blood 1950, 5 (3), 254−266. (39) Jaiswal, R. B.; Bhai, I.; Nath, N. Tyrosine crystals in bone marrow. Lancet 1968, 291 (7554), 1254−1255. (40) Constantinescu, M. B.; Chan, J. B.; Cassarino, D. S. Chondroid syringoma with tyrosine crystals: Case report and review of the literature. Amer. J. Dermopathol. 2010, 32 (2), 171−174. (41) Couce, M. E.; Perry, A.; Webb, P.; Kepes, J. J.; Scheithauer. Fibrous meningioma with tyrosine-rich crystals. Ultrastruct. Pathol. 1999, 23 (5), 341−345. (42) Ramachandran, E.; Natarajan, S. Crystal growth of some urinary stone constituents: I. In-vitro crystallization of L-tyrosine and its characterization. Cryst. Res. Technol. 2002, 37 (11), 1160−1164.

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Tyrosine Microcrystals Produced by Digestion of Proteins with

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