Leaf samples were collected from Texas Live Oak trees found on the Texas A & M International University campus. Five trees were selected to sample and represent the Texas Live Oak tree population (N27˚34'24.3" W99˚26'00.3"; N27˚34'23.4" W99˚26'12.3"; N27˚34'22.3", W99˚26'17.3"; N27˚34'34.5" W99˚25'49.0"; N27˚34'23.3" W99˚25'59.1"). Leaf samples collected were mature sized leaves that ranged from 8 centimeter to 12 centimeter in size. Leaf samples were stored in assigned labeled Ziploc™ bags placed in a −40˚C freezer or samples were immediately homogenized.

Leaves were washed, ground and homogenized using a Waring^® Laboratory variable-speed blender. Cold 0.01 M Tris-Cl buffer (0.15 M NaCl), pH 9.4 (1:8 w/v) was added and homogenized for one minute for each respective sample. Samples were stirred for one hour using a VWR^® Dyla-Dual™ Hot Plate Stirrer at 1000 rpm in a cold room (4˚C) to optimize homogenization. Then, the homogenate was filtered using cheesecloth and centrifuged at 8000 rpm at 10˚C for 30 minutes using an Avanti^® JE Centrifuge JA-20 Rotor. The crude extract was stored (at −40˚C) for later use or immediately used for agglutination assays.

Two-hundred microliter aliquot of blood samples including human (collected from Laredo Medical Center Laboratory), horse, rabbit and sheep (purchased from Biomérieux^® company) were mixed with 10 mL 0.01 M, phosphate buffer saline (PBS), pH 7.2 (0.15 M NaCl) in a 15 mL tube. The blood suspension was centrifuged at room temperature using a Hamilton Bell^® VanGuard V6500 Biohazard centrifuge with fixed speed of 3500 rpm for five minutes. At the end of centrifugation, the supernatant was discarded. A second wash or until the supernatant was clear to remove lysed red blood cells was done using 10 mL of 0.01 M, PBS. The pellet was dissolved with PBS to obtain a 2% blood suspension.

The crude extract and purified fractions were assayed for the presence of lectin activities using Corningware™ 96-well microtiter U-plates, 0.01 M PBS and 2% blood suspension. The sample was diluted by a serial two-fold dilution in PBS (50 μL) and incubated with a 2% suspension of RBCs (50 μL) at room temperature for an hour or until the negative control showed a red button formation. Agglutination activity was detected based on the RBCs appearance on the well; a positive result appears as a red-carpet layer, while negative results, appear as a red button in the bottom of the well.

The protein content of crude extract samples was determined with the Bradford method [39] using a QuickStart™ Bradford Protein Assay Kit.

The lectin from Q. fusiforms crude extracts was partially purified by one step chromatography using an ion-ex- change column chromatography with a Bio-Rad^® BioLogic™ low-pressure chromatography system. All absorbance readings were at 280 nm. The flow rate was 1 ml/min and collections of eluate with 3 mL/fraction. Twenty-six mL of Q. fusiformis crude extract was applied to DEAE-cellulose column, which was equilibrated and washed with Buffer A, 0.01 M Trizma-Cl with 0.15 M NaCl, pH 9.2. After washing, Buffer B, 0.01 M Trizma-Cl with 0.5 M NaCl , pH 9.2, was run into the column until absorbance was below < 0.01, then elution Buffer C, 0.01 M Trizma-Cl with 1.0 M NaCl, pH 9.2, was applied into the column. A Bio-Rad^® fraction collector

(#731 - 8304) was used to collect fractioned proteins. Fractioned proteins were pooled and dialyzed to remove the salts. Dialysis was performed with Thermo Scientific SnakeSkin^® dialysis tubing. The dialysate used was deionized water. The dialyzed samples were then freeze-dried.

High Performance Liquid Chromatography (HPLC) separation was performed on Prostar PS 220 system which has a UV detector and Rheodyne injector with 20 μl loop volume. Galaxie^© Software was applied for data collecting and processing. Purity assessment of the dialyzed and freeze-dried sample (2 μL of freeze-dried sample diluted in 100 μL deionized H₂O) was analyzed by HPLC. A Phenomenex RP-C18 (250 × 4.60 mm) column was used as the stationary phase. A gradient of 100% water to 100% methanol was used as mobile phase. The mobile phase was pumped at 1 ml/min and the eluents were monitored at 215 nm and 280 nm. The injection volumes of samples were 20 μl.

Data for blood group specificity and pH stability were obtained from five replicates. An analysis of variance (ANOVA) associated with a 5 × 4 factorial experiment in randomized complete blocks design was performed using the Statistical Analysis System (SAS 9.3)^® software. Mean comparisons were performed by way of a Bonferroni test using a type I error rate of 0.05. Data analysis for seasonal variation was carried out using a one-way ANOVA. Bivariate correlations among seasonal variation, soil moisture and pH were estimated and tested using a Pearson correlation analysis with level of significance for a two-tailed test set at 0.01.

The blood group specificity of Q. fusiformis lectin activity was investigated in four blood groups (horse, human, rabbit and sheep). The extracts of Q. fusiformis agglutinated to all blood groups tested, making Q. fusiformis a non-blood group specific lectin. Lectin specific activity (SA) was expressed as titer over milligrams of protein. For analysis, lectin specific activity was transformed to natural logarithm of specific activity (ln SA). ANOVA results reported a difference among the four blood groups tested, which was subjected to a Bonferroni test using a Type I error rate (α of 5%) for mean comparisons to determine significant differences (Table 1, Figure 2). The Bonferroni test showed a significant difference in the ln specific activity values of Q. fusiformis lectin between

^a Titer is the reciprocal of the lowest dilution that was positive for lectin activity. ^b Protein content was determined using Bradford assay. ^c Hemagglutination Activity (HA), is titer multiplied with sample volume (50 uL). ^d SA, Specific Activity is HA divided by the protein content. ^e ln Specific activity (SA), is the natural logarithm of specific activity. Values of ln SA are mean values from Bonferroni test using a type I error rate of 5%. ^f Blood groups horse, human and rabbit show no significant difference from each other. ^g Sheep blood group is significantly different from horse, human and rabbit.

*Significant (p<0.05) compared to horse, human and rabbit blood groups.

*Significant (p<0.05) compared to horse, human and rabbit blood groups.

Figure 2. Blood group specificity of Q. fusiformis lectin based on ln SA of mean comparisons analyzed with a Bonferroni test using a type I error rate of 5%.

sheep blood (ln SA = 8.30) and the three blood groups (horse, ln SA = 6.29), human (ln SA = 7.21) and rabbit (ln SA = 7.02). As shown in Figure 2, ln specific activity using sheep blood showed a significantly higher lectin activity as compared to the other three blood groups. However, lectinln specific activities of Q. fusiformis using horse, human and rabbit blood groups were not significantly different from each other.

Specificity is influenced by the limited number of contacts with carbohydrates and depth of the sugar binding sites [44] . In addition, any modification or substitution to a binding site can influence binding specificity [45] . The observed differences in Q. fusiformis lectin activity with different blood groups may be due to differences in carbohydrate-lectin binding interactions which can be attributed to differences in carbohydrates presented on the cell surfaces of the different blood groups.

The carbohydrates on the erythrocyte cellular surfaces are distinguishable among the four different blood groups [46] . There are reported monosaccharide determinants in the different blood groups, fucose in horse [47] , galactose in human and [47] -[49] and mannose in rabbit erythrocytes [50] . Horse, human and rabbit red blood cells may contain carbohydrate components on the cellular surface binding sites that are relatively less recognized by the Q. fusiformis lectin binding site. On the other hand, the carbohydrates found on the cellular surface of sheep red blood cells may contain carbohydrate units in a structure and position more specific and with higher affinity for the binding of Q. fusiformis lectin, subsequently increasing sheep erythrocyte agglutination. Moreover, lectin characteristics such as, multivalency may determine cross-linking interaction in binding recognition. Spatial distribution of multivalency among lectin structures may produce a higher level of specificity [51] [52] .

Quercus fusiformis lectin activity is similar to other plant lectins that are also non-blood group specific. Erythrina speciosa lectin was characterized as a non-blood group specific. Its lectin activity was examined in the human blood ABO system and animal blood groups, rabbit, mouse, sheep and horse [53] . Likewise, the blood group specificity for leaf lectin in Kalanochoe crenata was characterized as a non-blood group specific lectin, agglutinating to the different types of human blood red cells of the ABO system [36] . Artocarpus incisa seeds were also examined in a wide range of blood groups including human ABO system and animal blood groups, cow, goat, rabbit, pig and sheep. Artocarpus incisa seed lectin resulted in non-blood group specificity in humans ABO system, while rabbit blood group activity was not different from human ABO and the other four blood groups were significantly different from human and rabbit agglutination [54] . Similarly, Bryopsis plumosa lectin from a green marine alga agglutinated sheep and horse erythrocytes [55] . The blood group specificity of Q. fusiformis lectin activity suggests Q. fusiformis lectin is non-blood group specific with higher specificity directed towards sheep erythrocytes. The present study is the first to report the non-blood group specificity of the Q. fusiformis lectin.

Crude extracts of Q. fusiformis lectin were prepared and incubated in wide range of pH values with stirring for 24 hours at 4˚C. A Bonferroni test using a Type I error rate (α of 5%) was used to compare mean natural logarithm-transformed specific activity (ln SA) across pH levels (5.2, 6.2, 7.2, 8.2 and 9.2). Results indicated that Q. fusiformis lectin was stable at a pH range 5.2 to 8.2 while that for pH 9.2 was significantly lower than at pH 5.2 - 8.2 (Table 2, Figure 3). Different pH conditions were found to have profound effects on the tertiary and qua-

^a Titer is the reciprocal of the lowest dilution that was positive for lectin activity. ^b Protein content was determined using Bradford assay. ^c Hemagglutination Activity (HA), is titer multiplied with sample volume (50 μL). ^d SA, Specific Activity is HA divided by the protein content. ^e ln Specific activity (SA), is the natural logarithm of specific activity. Values of ln SA are mean values from Bonferroni test using a type I error rate of 5%. ^f pH levels not significant difference from each other, lectin activity was stable. ^g pH level significantly different from 5.2 - 9.2.

^*Significant, (p < 0.05) compared to pH range of 5.2 - 8.2.

^*Significant, (p < 0.05) compared to pH range of 5.2 - 8.2.

Figure 3. The effect of pH on Q. fusiformis lectin activity based on mean comparisons of ln SA analyzed with a Bonferroni test using a 5% type-I error rate.

ternary structure of proteins and can perturb protein conformational stability [36] [56] . Lectins have different optimum pH to maintain their stabilities [57] .

Literature reported that pH stabilities of plant lectins vary. For example, Morus rubra [58] and Ipomoea asarifolia [59] leaf lectin crude extracts were stable at pH 7.5, while the Korean mistletoe lectin crude extract was stable at pH 8.0 [60] . On the other hand, the pH stability of Kalanochoe crenata leaf lectin was at pH values 2 to 12 [36] . Kalanochoe crenata leaf lectin activity was stable from pH 2 to 7.5, and the activity was lost at a pH higher than 9.2 [36] . The effect of pH on lectin activity was studied in the leaves of Chorchorus olitours also in a broad range of pH values from pH 2.0 to 10.5. Chorchorus olitours exhibited a high agglutination activity from pH 7.2 to 8.0 with a dramatic decrease of lectin activity below acidic conditions of pH 6.5 and above basic pH of 9.0 [46] . The pH stability of Q. fusiformis lectin activity at a wide range of pH is quite similar to other leaf lectins.

Previous studies have reported lectin sources that display a seasonal variation in lectin activity [61] . For example, Elderberry (Sambucus nigra) and Black locust (Robinia pseudoacacia) bark lectins demonstrated a seasonal variation in content where lectin accumulation was higher during summer to winter compared to spring [42] . On the other hand, lectin activities in cultivated red alga (Kappaphycus alvarezii) varied with environmental characteristics such as heavy rain, solar radiation and low seawater temperatures [61] . It was hypothesized that lectin fluctuation found in both Elderberry and Black locust barks as contributed by seasonal variation resembles the behavior of plant storage proteins [42] . Although, many plant lectins mimic the behavior of plant storage proteins, these lectins should not be classified as storage proteins [2] . Costa et al. [43] studied Phthirusa pyrilfolia leaf lectin activity with an activity higher during sunny weather. This was attributed to mistletoe’s slower rate of photosynthesis promoting high lectin production.

In this study, Q. fusiformis leaf samples were collected during the fall, winter and summer seasons and lectin activities were compared. The mean ln SA (specific activity) was 7.6, 7.8 and 8.2 for fall, winter and summer, respectively (Table 3). Results of the correlation analysis determined that there was no significant correlation in mean ln SA for Q. fusiformis lectin during the different seasons, fall, winter and summer (Figure 4). Compared to previous studies, this study has shown that Q. fusiformis lectin activity was not affected by seasonal variations.

Since the productivity of plants including biomolecule synthesis is affected by environmental factors, soil properties, moisture and pH were examined to determine if any of these factors affect lectin activity in Q. fusiformis. To date, the literature with regards to soil properties namely, moisture and pH affecting lectin activity are scarce. The lectin activity of common beans (Phaseolus vulgaris) was examined in three different soil types in a semiarid region. The result of this study reported differences in lectin concentration and suggested environmental factors to contribute to lectin difference [62] . Results of the present study showed the average soil pH reading for 20 - 30 centimeter depth in fall was pH 8.36 and in winter was pH 8.31. On the other hand, the average moisture loss for the 20 - 30 centimeter depth in fall was 3% and for winter 5.6%.

The soil moisture and pH were subjected to a correlation analysis with level of significance for a two-tailed test at level 0.01 as shown in Table 4. Soil moisture and pH reflect the chemical status of soil conditions for the Q. fusiformis. Laredo soil is usually alkaline throughout [63] . Since the pH results of soil are basic, this may indicate that there may be some nutrients unavailable to Q. fusiformis, the production and expression of lectin may be affected. On the other hand, Q. fusiformis is a drought-tolerant tree and only requires water every three to

^a Titer is the reciprocal of the lowest dilution that was positive for lectin activity. ^b Protein content was determined using Bradford assay. ^c Hemagglutination Activity (HA), is titer multiplied with sample volume (50 uL). ^d SA, Specific Activity is HA divided by the protein content. ^e ln Specific activity (SA), is the natural logarithm of specific activity. Values of ln SA are mean values from one-way ANOVA, t-test.

^**Correlation is significant at the level 0.01 (2-tailed).

four weeks. Analysis of the results for soil moisture and soil pH showed no significant correlation with ln SA for Q. fusiformis lectin.

There are two physiological proposed roles for plant lectins; the first is in plant defense mechanisms and the second, symbiotic nitrogen-fixing bacteria association [7] [64] [65] . Plant lectins have been investigated for their resistance to insects [66] , bacteria [1] [27] [67] [68] and fungi [1] [2] [69] [70] . The study of Q. fusiformis lectin is relevant since new compounds and especially plant lectins with antifungal activity are of high interest due to rising fungal resistance to common fungicides. There are only a few plant lectins reported to have antifungal activities.

The antifungal activity of Q. fusiformis lectin was tested against Aspergillus niger and Rhizopus stolonifer. These two fungi are common pathogens in fruits and vegetables and cause rot disease [71] . Freeze-dried crude extracts of 125 μg/μl, 250 µg/µl and 500 μg/μl concentrations did not show any antifungal activity against A. niger and R. stolonifer. Similarly, the knife bean (Canavalia gladiata) lectin, was devoid of antifungal activity when tested against three fungi Botrytis cinerea, Myocospharella arachidcola and Fusarium oxysporum [72] . In contrast, plant mistletoe (Phthirusapyrfolia) lectin was tested against eleven fungi (Aspergillus niger, A. flavus, A. fumigatus, Rhizopus arrhizue, Paeciloyces variottie, Fusarium moniliforme, F. laterituium, Candida albicans, C. burneses, C. tropicalis, C. parapsilosis, Saccharomyces cerevisiae and Rhizoctnia solani) and demonstrated antifungal activity against two fungi F. lateritium and R. solani [43] . Red kidney bean (Phaseolus vulgaris)lectin reported by Ye et al. [73] , also expressed antifungal activity but towards three fungal species, Fusarium oxysporum, Rhizoctonia solani and Coprinus comatus.

The crude extraction of lectin from Q. fusiformis was achieved by homogenization in Tris-Cl, pH 9.2 (0.15 M NaCl) buffer and stirred at 4˚C for one hour. Partial purification of Q. fusiformis crude extract was carried out by a one-step column chromatography technique using the ion-exchange chromatography column. The weak anionic DEAE-cellulose column (Pall Life Sciences AcroSep™, Product No. 20067-C001) consisted of a matrix with positive charges that will bind negatively charged proteins. Crude extracts of Q. fusiformis at pH 9.2 ran through the DEAE column chromatography resulted in the binding of proteins. The chromatography profile showed three peaks, peaks are labeled with abbreviation letter D to indicate DEAE. As shown in Figure 5, the Q. fusiformis crude extract resulted in the separation of proteins that corresponded to 3 peaks. The first peak (D1), the major peak, contained unbound proteins. The second peak (D2) and third peak (D3) corresponded to bound proteins that were eluted from the DEAE column using half stepwise salt concentration gradient 0.5 and 1.0M, respectively.

Quercus fusiformis DEAE fractions, D1, D2 and D3 were tested for agglutination activities. These fractions did not exhibit any agglutinating activity. The absence of lectin activity in DEAE fractions may be for two reasons. First, fractions may not contain enough lectin concentration to have an activity detectable by the sensitivity of the agglutination assay due to dilution effect of column chromatography. Secondly, the high salt concentration used to elute proteins in peaks D2 and D3 may indicate an effect on conformational structure and stability of the lectin. Thus, fractions from each peak were pooled, dialyzed and lyophilized. Dialyzed samples were measured for pH with litmus paper and demonstrated a neutral pH.

Lectin activity is affected when pH conditions are modified for basic or acidic conditions [57] . This is true for Q. fusiformis lectin activity, and provides support for the effect of pH results when lectin activity decrease at pH level 9.2. The lyophilized samples were diluted with 200 µl of millipore water and subjected to agglutination assays to detect presence of lectin activity. Table 5 shows results of the agglutination assays. Results showed that all peaks contained lectin activity. The binding of Q. fusiformis lectin to the DEAE column could be attributed to the lectin amino acid composition. It can be assumed that Q. fusiformis lectin has a predominance of negatively charged aspartic acid and/or glutamic acid residues. These amino acids would give the lectin a net negative charge at the basic pH due to the R-carboxylic groups (COO^-) present. Thus, the negatively charged Q. fusiformis lectins bind to the positive charged matrix of DEAE. For example, red marine alga (Vidalia obtusiloba C.) lectin was purified through two chromatographic steps, with its first step being an ion exchange DEAE- cellulose column. This lectin was reported to be rich in aspartic acid, glutamic acid and leucine [74] .

The first peak, D1 contained all unbound proteins including unbound lectins, thus D1 was not subjected to purity determination. Between D2 and D3, D2 was chosen for purity determination owing to the relatively higher protein concentration as well as higher lectin activity observed in peak 2, D2. The purity of sample D2 was assessed using high performance liquid chromatography (HPLC). Based on the analytical assessment of D2, Q. fusiformis

lectin is either only partially purified or D2 contains at least three isolectins since 3 peaks (retention times, 1.0, 1.5 and 2.5 min) were obtained after HPLC (Figure 6). Isolectins are defined as closely related lectins difficult to separate since they have a similar amino acid composition.