Phosphoglucomutase isoenzymes in pollen grains

Phosphoglucomutase isoenzymes in pollen grains

SCIENTIFIC & TECHNICAL Phosphoglucomutase isoenzymes in pollen grains P DONNELLY," K FRYER Wetherby Laboratory, Forensic Science Service, Sandbeck Wa...

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Phosphoglucomutase isoenzymes in pollen grains P DONNELLY," K FRYER Wetherby Laboratory, Forensic Science Service, Sandbeck Way, Audby Lane, Wetherby, West Yorkshire, United Kingdom LS22 4DN

and CA SMITH? Department of Biological Sciences, t h e Manchester Metropolitan University, Manchester, United Kingdom M1 5GD Journal of the Forensic Science Society 1993; 33: 153-157 Received 30 April 1992; accepted 11 December 1992 The possibility that contaminating pollen grains interfere with the electrophoretic phosphoglucomutase phenotyping of human body fluid stained samples was investigated. Pollen grains from forty-two different species were examined. Forty showed detectable amounts of activities of isoenzymes of phosphoglucomutase following starch gel electro~horesis. while 23 showed discernible isoenzymes followihg ultrathin isoelectric focussing. In several cases, phosphoglucomutases extracted from pollen grains comigrated with human types. Phosphoglucomutase isoenzymes of pollen grains of Sambucus nigra (common elder), in particular, interfered with phosphoglucomutase phenotyping using starch gel electrophoresis and isoelectric focussing. These results suggest that it may be necessary to examine some forensic specimens for contamination with pollen prior to the electrophoretic separation of phosphoglucomutase isoenzymes.

Die Miiglichkeit, dass Elektrophorese-PhosphoglucomutaseTv~isierung von Kor~erfliissigkeit-Trockens~urendurch ~ d l e n - ~ o z a m i n a t i o6eeinfluslt n wird, wurde' naher untersucht: 40 von 42 untersuchten Pollen-Spezies zeigten nachweisbare Isoenzym-Aktivitaten von Phosphoglucomutase nach Starkegel-Elektro~horese:bei 23 Proben i a r e n Isoenzyme nach iltradiinne; isoelektrische Fokussierung nachweisbar. In mehreren Fallen kam es zu einer C O - ~ G r a t i o nder Phosphoglucomutase von Pollen mit jener menschlicher Provenienz. Speziell die Phosphoglucomutase Isoenzyme der Pollen von Holunder beeintrachtigten die Phosphoglucomutase-Typisierung bei der Verwendung von Starkegel-Elektrophorese und isoelektrischer Fokussierung. Die Ergebnisse weisen darauf hin, dass bei der forensischen Untersuchung von Proben mittels Elektrophorese-Trennung der Phospho lucomutase Isoenzyme diew vorgangig auf Kontamination Jurch Pollen zu untersuchen sind.

La possibilitk qu'une contamination par des grains de pollen interfbre avec la determination du phCnotype du phosphoglucomutase par Clectrophori%e de liquide biologique humain a CtC CtudiCe. Les grains de pollen de 42 espi.ces diffirentes ont CtC examinks. 40 d'entre eux ont montrC des quantitCs dktectables dlactivitC d'isoenzymes de phosphoglucomutase a p r b Clectrophorkse sur gel d'amidon, alors que 23 ont montrC de tels isoenzymes lors de I'utilisation de focalisation isoClectrique ultra-mince. Dans plusieurs cas les phosphoglucomutases extraites des grains de pollen migraient en m&me temps que les types humains. Les isoenzymes du phosphoglucomutase de grains de pollen de Sambucus nigra (sureau noir), en particulier, interf2rent avec la determination du phknotype phosphoglucomutase en utilisant I'Clectrophorbse sur gel d'amidon et la focalisation isoklectrique. Ces rCsultats sugghent qu'il peut &tre nCcessaire d'examiner les spCcimens forensiques pour une contamination par des pollens avant de proceder 5 la skparation ClectrophorCtique des isoenzymes phosphoglucomutases.

Se investiga la posibilidad de que granos de polen contaminantes, interfieran en el tipaje por electroforesis de la fosfoglucomutasa en muestras de manchas de fluidos biol6gic6s humanos. Se examinan granos de polen de cuarenta v dos es~ecies diferentes. Cuarenta de ellas mostraron'cantidade's detectables de actividades de isoenzimas de fosfoglucomutasa tras electroforesis en gel de almidon. mientras aue 23 mostraron isoenzimas discernibles tras isoelectroenfoqhe en geles ultrafinos. En algunos casos, las fosfoglucomutasas extraidas de granos de polen co-migraban con tipos humanos. Las isoenzimas de fosfoglucomutasa de granos de polen de Sambucus nigra (Sauco comun), en particular, interfieren en el tipaje de fosfoglucomutasa usando electroforesis de gel de almidon e isoelectroenfoque. Estos resultados sugieren que puede ser necesario investigar la contaminaci6n con polen en algunos especimenes forenses antes de la separaci6n por electroforesis de las isoenzimas de fosfoglucomutasa. ~

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Key Words: Phosphoglucomutase; Starch gel electrophoresis; Ultrathin isoelectric focusing; Pollen grains; Contamination. *Present address: The Scenes of Crime Department, West Yorkshire Police, Brotherton House, Leeds LSl 2RD.

t Corresponding author. JFSS 1993; 33(3): 153-157

Introduction The separation of isoenzymes of phosphoglucomutase (PGM), E.C. by starch gel electrophoresis (SGE) and isoelectric focusing (IEF) is frequently used in the forensic sciences as a discriminating procedure [I-31. Spencer [2] showed that individuals could be divided into three phenotypes on the basis of differences in their respective isoenzymes of PGM, as separated by SGE. These phenotypes are designated PGMl, PGM2 and PGM2 - 1. The application of IEF to PGM phenotyping by Bark et al. [3] led to a refinement of these phenotypes. Thus the PGMl group was found to consist of three phenotypes designated PGMl+ , PGM1-, and PGMl + 1. Similarly, PGM2 consists of PGM2+, PGM2- and PGM2 + 2-, while PGM2 - 1 subdivides into four groups: PGM2 + 1+ , PGM2 + 1- , PGM2 - 1+ and PGM2 - I-. The presence of these 10 phenotypes in the population can be explained by assuming the presence of four alleles, PGM1+,PGM1-, pGM2+ and PGM2-, at the PGM locus [3]. PGM phenotyping of body fluid stained samples using electrophoretic procedures is now routine in forensic laboratories. However, an investigation at the Wetherby Forensic Science Laboratory of a semenstained sample gave anomalous results. Following SGE, the highest PGM activity was associated with an isoenzyme indicative of PGMl phenotype, although the distribution of isoenzymes was diagnostic of a PGM2 - 1 phenotype. I E F phenotyping of the stain was unsuccessful because of the limited amount of biological material available. A microscopic examination of the material showed the presence of large numbers of pollen grains, and the possibility that the grains were interfering with the staining procedures for PGM was therefore noted. Phosphoglucomutase activity is known in plants tissues, including pollen grains [4, 51, suggesting that pollen grain contamination of body fluid stained samples may cause problems when identifying PGM phenotypes using electrophoretic procedures. The reported study was initiated to determine the extent of this potential problem.

Methods Pollen grains were collected by gently brushing the grains into polythene bags using a fine paintbrush. Collections were performed in the Wetherby area during May-August. The presence of PGM activity in the grains was estimated by adding 2 mg of pollen grains to 1.0 ml of PGM buffer, as described by Culliford [I], in a polythene centrifuge tube. After soaking for 4 hours

at 4°C the grains were sedimented by centrifugation and discarded. The supernant was stored at -18OC prior to further examination. Phosphoglucomutase activity was determined by mixing 0.5 ml of the extract with 0.5 ml of the substrate reagent described by Culliford [I] followed by a 15-minute incubation at 37°C. Activity was assessed visually by reference to the colour developed using distilled water (negative control) and extracts of human blood (positive control) in place of the pollen grain extract. Pollen grain extracts showing PGM activity were subjected to SGE (after Spencer et al. [2]), using 1mm thick gels at 6.5 V/cm for 16 h at 4°C [I], and ultrathin IEF in 0.15 mm thick polyacrylamide gels, initially using gradients of 3.5-10.0 p H divisions [3]. Extracts of pollen with isoenzymes of similar isoelectric points to human phenotypes were refocused with gradients of




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FIGURE 1 Electrophoretic separations of PGM isoenzymes shown schematically. (a) Starch gel electrophoresis of PGM isoenzymes from pollen grains (PG) of Sambucus nigra (common elder) and human phenotypes PGM1, PGM2 and PGM2 - 1; (b) ultrathin isoelectric focusing of extracts of pollen from S nigm, pollen mixed with human semen of phenotype PGMl+ and human semen of phenotypes PGMl+ and PGM2+ l+. Note in both (a) and (b), the overlapping banding patterns between some of the lanes gives the possibility of ambiguous interpretations of samples contaminated with pollen.

JFSS 1993; 33(3): 153-157

5-0-7-0pH divisions. All I E F was performed at 3000 Vh with starting conditions of 2000 V and 5 mA (i.e., 4WE) and at 4°C. The pollen grain extracts were electrophoresed and isoelectrically focused as individual samples and also, in some cases, following mixing with extracts of human blood or semen of known PGM phenotype (Figure 1). The isoelectric

points of PGM isoenzymes were determined by comparison with proteins of known isoelectric point (Pharmacia, Milton Keynes, UK). Results and discussion Table 1 summarises the results of the investigation. Phosphoglucomutase activity was investigated in

TABLE 1 Investigation of PGM isoenzyme activity in pollen grains by starch gel electrophoresis and isoelectric focusing Species Human blood

Allium schoenoprasum (Chives) Begonia sp Bellis perennis (Daisy) Briza sp (Quaking grass) Buddleia sp

Calystegia sy lvatica (Greater bindweed) Campanula sp (Canterbury bell) Chamaenerian angustifolium (Rosebay) Crataegus sp (Hawthorn) Crocus sp Cydonis japonica (Japanese quince) Dactylis glomerata (Cocksfoot) Dianthus sp (Carnation) Digitalis purpurea (Foxglove)

Eccrernocarpus scaber (Chilean glory flower) Endymion non-scriptus (Bluebell) Epilobium hirsutum (Great willow herb) Eremurus sp (Foxtail) Fuchsia sp

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IEF ( p l values)


PGM1, PGM2, PGM2 -

+ + +++ + ++

Isoenzymes anodal to human types Isoenzymes anodal to human types Isoenzymes anodal to human types ND


Isoenzymes anodal to human types

+ +

Isoenzymes anodal to human types Isoenzymes anodal to human types

+++ +

Isoenzymes anodal to human types ND

v strong

++ ++

Isoenzymes anodal to human types Isoenzymes anodal to human types

v strong

+ ++ ++ +

Isoenzymes anodal to human types Isoenzymes anodal to human types ND

Isoenzymes anodal to human types

(a - (I-)) (a + (I+)) (b - (2-1) (b + (2+)) v weak weak weak weak

v weak v weak v weak v weak v strong weak weak weak strong strong

weak weak v strong strong v strong v strong

weak weak

TABLE 1 (Continued) -

Species Helianthum sp (Rockrose) Holcus lanatus (Yorkshire fog) Hypericum sp (St John's wort) Imputiens sp (Balsam) Iris siberica

Laburnum sp Lathyrus ordoratus (Sweetpea) Lavandula spica (Lavender) Leycestria formosca (Himalayan honeysuckle) Lilium sp (Lily) Lonicera japonica (Honeysuckle) Lupinus sp (Lupin) Montbretia sp Papaver sp (POPPY) Ranunculus sp (Buttercup) Rosa canina (Dog rose) Sambucus nigra (Common elder)

Solanum melongen (Aubergine) Syringa vulgaris (Lilac) Tagetes patula (French marigold) Tropaelium majus (Nasturtium) Urtica diolca (Stinging nettle) Vicia sativa (Common vetch)


+ +

+ + +++ + + ND

+ +++

+ + +


SGE Isoenzymes anodal to human types Isoenzymes anodal to human types Isoenzymes anodal to human types Isoenzymes anodal to human types Isoenzymes anodal to human types

Isoenzymes anodal to human types Isoenzymes anodal to human types NT Isoenzymes anodal to human types Isoenzymes anodal to human types Isoenzymes anodal to human types Isoenzymes anodal to human types ND Isoenzymes anodal to human types

++ ++ +++

Isoenzymes anodal to human types ND

+ ++

Isoenzymes anodal to human types


Isoenzymes anodal to human types

+ + +

Isoenzymes anodal to human types ND


Isoenzymes co-migrate with some of PGM1, PGM2 and PGM2 - 1

IEF ( P I values)



5.20 4-95 5.90* 5.25 5.15 4.90 4.75

v weak strong v strong v strong v strong v strong v strong

5.40 4.85 NT

v weak weak





ND 5.40 5.35 4.90 ND

v strong v strong v weak

5-90* 5.65 5.35 4.60 6-45* 6.25* 5-35 5.05 4.80 4.60 ND

v strong v strong v strong weak v strong v strong v strong strong strong strong

Isoenzymes anodal to human types NT


= comparable to human blood PGM activity range from + = low to + * isoelectric point similar in value to a human PGM isoenzyme ND activity not detected NT not tested

JFSS 1993; 33(3): 153-157

pollen grains collected from 42 different plant species. Extracts from only two types failed to show PGM activity. Weedon and Gottlieb [5] have emphasised that an apparent absence of enzyme activity need not necessarily indicate a lack of enzyme in the grain, since the enzyme may be present, but in a non-leachable form. Further, the age of the plant, its nutritional status and environmental conditions during pollen grain development all affect the amounts of PGM activity [5]. Thus the relative proportions of PGM activities listed in Table 1 cannot be regarded as fixed. The results of SGE are not presented in detail, but phosphoglucomutase isoenzymes were detected in 27 of the 42 extracts subjected to SGE. These generally appeared anodic to the human isoenzymes and were therefore faster migrating. Thus the presence of pollen grains should not normally present a problem when PGM typing human biological material using SGE. However, an exception to this general situation occurs with Sarnbucus nigra (common elder) since pollen grain extracts showed strong staining in regions of the gel associated with all three PGM phenotypes as separated by SGE (Figure 1). Pollen grains not investigated may also, of course, co-migrate with human isoenzymes. The presence of PGM isoenzymes of measurable isoelectric points was detected in 23 of the pollen grain extracts subjected to ultrathin IEF. The isoelectric points showed a range of values which in several cases were comparable to those of human PGM isoenzymes (Table 1). Isoenzymes separated from pollen grains of common elder (S. nigra) were particularly noteworthy in this respect. Ultrathin IEF of mixtures of extracts of common elder pollen with human blood or semen of known PGM1+ phenotype (results not listed in detail) gave isoenzyme patterns

JFSS 1993; 33(3): 153-157

which strongly resembled that of a PGM2+ 1+ phenotype (Figure 1). Further, the large variation in the isoelectric points of the pollen PGM isoenzymes investigated suggests that pollen grains in other plant species may present additional problems to forensic PGM phenotyping. The present study was restricted to pollen grains from a number of plant species. Isoenzymes of PGM appear widely distributed among pollen grain types. Contamination of forensic samples by some pollen grains may therefore pose a serious problem to PGM phenotyping, particularly if I E F is the method of choice. It may also be noted that this contamination may not be apparent, given that the size of pollen grains varies appreciably between species, e.g. from about 200 to over 10 000 per milligram weight. Our conclusions suggest that in some instances it may be necessary to examine forensic specimens for possible contamination with pollen grains, prior to their PGM phenotyping using SGE and IEF.

References 1. Culliford BJ. The examination and typing of bloodstains in the Crime Laboratory, USA: The U.S. Department of Justice Law Enforcement Assistance Administration, 1971: 122-123. 2. Spencer N, Hopkinson D A and Harris H. Phosphoglucomutase polymorphism in man. Nature 1964; 204: 742-745. 3. Bark JE, Harris MJ, and Firth M. Typing of the common PGM variants using isoelectric focusing: a new interpretation of the PGM system. Journal of the Forensic Science Society 1976; 16: 115-120. 4. Dickinson DB and Davies MD. Pollen development and physiology, London: Butterworths, 1971: 190-193. 5. Weedon NF, and Gottlieb LD. Distinguishing allozymes and isoenzymes of phosphoglucomutase by electrophoretic comparisons of pollen and somatic tissues. Biochemical Genetics 1979; 19: 287-296.