Hum. Genet. 34, 53 56 (1976) © by Springer-Verlag 1976

Rapid Detection of Glyoxalase I (GLO) on Cellulose Acetate Gel and the Distribution of GLO Variants in a Dutch Population P. Meera K h a n a n d B i a n c a A n n e m a r i e D o p p e r t Department of Human Genetics, University of Leiden Received February 16, 1976

Summary. A rapid eleetrophoretie procedure is described for detecting the human red cell glyoxalase I variants (GLO 1, GLO 2--1, and GLO 2) on cellulose acetate gel (cellogel) on which the sites of enzymed activity are visualized as purple bands against white background. The frequency of GLO ~ gene in a Dutch population living in and around Leiden was found ~o be 0.4544.

Introduction

K S m p f et al. (1975a, b) h a v e described a m e t h o d for d e t e c t i n g g l y o x a l a s e I (GLO; E.C. : 4.4.1.5) on s t a r c h gel a n d discovered t h e h u m a n r e d cell GLO polym o r p h i s m d e t e r m i n e d b y a p a i r of alleles (GLO 1 a n d G L O ~) occurring at an autosomal locus. The corresponding p h e n o t y p e s were called GLO 1, GLO 2 - - 1 , a n d GLO 2. The incidence of G L O 1 gene in a s o u t h w e s t e r n G e r m a n p o p u l a t i o n was r e p o r t e d to be 0.427 ( K 6 m p f a n d Bissbort, 1975). More recently, t h e locus for GLO was f o u n d to be l i n k e d to t h e H L A region (Lewis et al., 1976; W e i t k a m p et al., 1976; Mcera K h a n et al., 1976). A s t u d y on t h e segregation p a t t e r n s of G L O v a r i a n t s in a p a n e l of 11 families w i t h e s t a b l i s h e d r e c o m b i n a t i o n s b e t w e e n t h e genes in t h e m a j o r h i s t o e o m p a t i b i l i t y complex (Bijnen et M., 1976) has i n d i c a t e d t h a t t h e G L O is s i t u a t e d b e t w e e n t h e H L A region a n d P G M s on chromosome 6 of m a n (Meera K h a n et al., 1976). The aim of this p a p e r is to describe a r a p i d electrophoretic m e t h o d for d e t e c t i n g t h e GLO a c t i v i t y on eellogel a n d to r e p o r t t h e incidence of GLO v a r i a n t s in a Dutch population. Material and Methods

Blood samples were eelleeted from 757 unrelated Dutch donors living in and around Leiden. Hemotysates with a hemoglobin concentration of about 10 g/100 ml were prepared (Meera Khan and Ruttazzi, 1968) using the "lysis buffer" (Meera Khan, 1971) without DFP, and stored routinely in liquid nitrogen. The general procedure for enzyme electrophoresis on eellogeI reported by Meera Khan (1971) was followed throughout. Therefore, only the features peculiar to GLO are described below. Electrophoresis. Buffer system: (0.03M) tris (0.03M) barbiturie acid pH 8.0 containing 0.2 ml of 1~ fi-mercaptoethanoi and 0.4 ml of 1M MgCI~/1; sample application: about 2 B1of hemolysate on the eathodal side of the gel; initial voltage: 200 V at constant current ; duration: 3 h at room temperature.

54

P. Meera Khan and B. A. Doppert

Fig. 1. Electropherogram showing human red cell GLO patterns on cellogel. Channels 1, 2, 3, and 4 carry phenotypes 1, 2, 2--1, and 1 respectively. Hemoglobin A (Hb A) is seen in between the origin and GLO. Minor band migrating cathodal to origin in each of the 4 channels is Hb A 2

Staining. It is done in two successive steps using two different reaction solutions. Solution I: 1.6 ml 0.1M phosphate buffer pH 6.5, 100 B1 methyl glyoxal (MG) (Sigma), 12 mg reduced glutathione (GSH) (Boehringer), and 0.4 ml of 2 mg/ml solution of MTT tetrazolium (MTT) (Sigma). Solution II: 1.8 ml 0.1M tris-HC1 pH 7.8 and 0.2 ml of 2 mg/ml solution of 2,6dichlorophenolindophenol-sodium (DCIP) (Merck). After electrophoresis the gel (16 em x 17 cm X 500 ~z) is treated first with freshly prepared solution I. The gel is then blotted just to remove the excess of reaction mixture and treated almost immediately with the second solution. An uneven blue to purple patches are seen all over the gel. But when it is left in a moist chamber at room temperature, purple bands appear at the site of GLO activity while the uneven color in the background gradually disappears (Fig. 1). I t may be noted that K6mpf et al. (1975a) observed "colorless bands against a blue background" in their starch gel procedure. This reversal appears to be pH-dependent, because, at pH levels higher than 8.0 white bands against blue background appear also on celloge]. An omission of MG, GSH, MTT, or DCIP in the reaction mixtures prevents the formation of these bands. The bands usually appear in about 15 min of incubation. For about 1 h they can be seen very clearly but after a few hours the purple bands turn pale while the background becomes blue in about 48 h. I f a more dilute electrophoretic buffer, for example, 0.02M in stead of 0.03M is used, the bands appear in about 11/2 h and stay prominently for about 10 h and fade away in another 10 h. In a 0.04M buffer on the other hand, sharp bands appear within l0 rain but fade away in less than 1 h. The bands, however, can be made to stay colored longer by fixing the gels in formaldehyde and washing off the reactants present in the background with demineralized water. Results and Discussion T h e e l e c t r o p h o r e t i e p a t t e r n s o f t h e p h e n o t y p e s G L O 1, G L O 2 - - 1 , a n d G L O 2 o n cellogel are seen in F i g u r e 1 : t h e s e are c o m p a r a b l e to t h o s e r e p o r t e d b y K 6 m p f et al. (1975a). T h e t y p i c a l t r i p l e - b a n d e d p a t t e r n o f t h e h e t e r o z y g o t e c o m p a r e d to t h e s i n g l e - b a n d e d p a t t e r n s of b o t h t h e h o m o z y g o t e s s u g g e s t s t h a t t h e f u n c t i o n a l

GLO Variation in a Dutch Population

55

Table 1 Distribution of phenotypes and alleles of red cell GLO in populations of Leiden and T/ibingen Population (N)

Phenotypes

Leidena (757) Tiibingenb (655)

obs. exp. obs. exp.

Z~ GLO 1

GLO 2--1

158 372 ( 1 5 6 . 3 1 ) (375.35) 114 331 ( 1 1 9 . 2 6 ) (320.46)

Gene frequency

GLO 2 227 (225.34) 210 (215.28)

GLO 1 : 0.4544 GL02:0.5456 GLOi: 0.4267 GL02:0.5733

0.0604 0.7082

a This report. b KSmpf and Bissbort (1975). Table 2. Segregation patterns of GLO phenotypes in 41 Dutch families Mating type

1 1 2 1 2 2--1 Total

× × × × × ×

1 2 2 2--1 2--1 2--1

No. of familiesa, b

No. of offspring b 1

2

1 (1.75p 4 (5.04) 6 (3.63) 9 (8.40) 15(12.10) 6(10.08)

4 --15(17) -1(4)

41

20

1

2

total

-20 -19(17) 27(29.5) 10(8)

--23 -32(29.5) 5(4)

4 20 23 34 59 16

76

60

d~

gz

1 1 2

0.4706 0.4237 3.0000

156

a Figures within brackets indicate the expected values calculated, based on the hypothesis that the GLO variation in this population is determined by a pair of alleles occurring at an autosomal locus and taking the GLO 1 frequency as 0.4544 (Table 1). b The observed and expected values of the parental matings differ only by chance 01~ = 4.4727 ; 0.4 < P > 0.5). This is also true for the values of the children's phenotypes. molecule of GLO is p r o b a b l y a dimer. No rare v a r i a n t s were encountered in the present population. The G L O 1 allele appears to be more f r e q u e n t i n the D u t c h p o p u l a t i o n t h a n in the people living in the s o u t h w e s t e r n p a r t of G e r m a n y ( K 6 m p f a n d Bissbort, 1975) b u t the difference is n o t significant (F = 2.208 with 1 d / ; 0.10 < P1 < 0.15) (Table 1). The conclusions 1that can be d r a w n from the results presented in Tables 1 a n d 2 agree with those of K 6 m p f et al. (1975a, b) a n d s u p p o r t the hypothesis t h a t the electrophoretically detectable v a r i a t i o n of red cell glyoxalase I is d e t e r m i n e d b y a pair of alleles occurring a t a n autosomal locus i n man. A n a p p l i c a t i o n of the H a r d y - W e i n b e r g law to the d a t a in Table 1 d e m o n s t r a t e s t h a t the genetically d e t e r m i n e d v a r i a n t s of GLO exist in perfect equilibrium in b o t h the populations so far investigated. I t appears, therefore, t h a t (in spite of the fact t h a t G L O is linked to H L A region a n d t h a t the glyoxalase system has been implicated in the regulation of red cell division a n d t h u s in carcinogenesis) (Szent-GySrgyi et al., 1967), there is no obvious preferential selection for one or the other of the three genotypes of GLO e n c o u n t e r e d in these populations.

56

P. Meera Khan and B. A. Doppert

Acknowledgements. We are grateful to Dr. J. G. Eernisse, the director of the Netherlands Red Cross Blood Transfusion Center, Leiden, for permitting us to use the donor sample and to Mrs. C. H. van Nouhuys-Maas for help in collecting the family material, to Miss L. M. M. Wijnen and Mrs. A. C. Ebeli-Struyk for assistance in the laboratory, and to Miss H. M. Oostvriesland for preparing this manuscript. The work has been supported by grants from the Netherlands Foundation for Medical Research (Nos. 13-23-11 and 13-41-04) and from the National Institutes of Health of the United States of America (Contract No. NO1-CB-43 940).

References Bijnen, A. B., Schreuder, I., Meera Khan, P., Allen, F. H., Giles, C. M., Los, W. R. T., Volkers, W. S., van Rood, J. J. : Linkage relationships of the loci of the major histoeompatibility complex in families with a recombination in the HLA region. J. Immunogenet. (in press, 1976) Lewis, M., Kaita, H., Chown, B., Bowen, P., Lee, C. S. N., McDonald, S., Giblett, E. R., Anderson, J., Dossetor, J. B., Schlaut, J., Pal, K. R. M., Singal, D. P., Steinberg, A. G. : A genetic linkage analysis of chromosome 6 markers Chide, HLA and glyoxalase. In: Proceedings of the Third International Conference on Human Gene Mapping, Baltimore (1975). Birth Defects: Original Article Series (The National Foundation, New York) (in press, 1976) KSmpf, J., Bissbort, S. : Population genetics of red cell glyoxalase I (E. C. : 4.4.1.5). Gene frequencies in south-western Germany. Kumangenetik 28, 175--176 (1975) K6mpL J., Bissbort, S., Gussmann, S., Ritter, H.: Polymorphism of red cell glyoxalase I (E.C. : 4.4.1.5). A new genetic marker in man. Humangenetik 27, 141--143 (1975a) KSmpf, J., Bissbort, S., Ritter, H. : Red cell glyoxalase I (E.C. : 4.4.1.5): formal genetics and linkage relations. Humangenetik 28, 249--251 (1975b) Meera Khan, P.: Enzyme electrophoresis on cellulose acetate gel. Arch. Bioehem. Biophys. 145,470--483 (1971) Meera Khan, P., Rattazzi, M. C. : Rapid detection of 6-phosphogluconate dehydrogenase variants by electrophoresis on cellulose acetate gel. Biochem. Genet. 2, 231--235 (1968) Meera Khan, P., Volkers, W. S., Doppert, B. A., Bijnen, A. B., Schreuder, I., van Rood, J. J. : The locus for glyoxalase I (GLO) is between H L A - A and P G M 3 on chromosome 6 of man. In: Proceedings of the Third International Conference on Human Gene Mapping, Baltimore (1975). Birth Defects: Original Article Series (The National Foundation, New York) (in press, 1976) Szent-GySrgyi, A., Egyfid, L. G., McLaughlin, J. A.: Keto-aldehydes and cell division. Glyoxal derivatives may be regulators of cell division and open a new approach to cancer. Science 155, 539--541 (1967) Weitkamp, L. R., Guttormsen, S. A. : Genetic linkage of a locus for erythrocyte glyoxalase (GLO) with HLA and Bf. In: Proceedings of the Third International Conference on Human Gene Mapping, Baltimore (1975). Birth Defects: Original Article Series (The National Foundation, New York) (in press, 1976) Dr. P. Meera Khan Department of Human Genetics, RUL Wassenaarseweg 72 Leiden, Netherlands