A story of enzymological serendipity

The eggs were fertile – the embryos developed initially normally – but they died in the egg at about day 10.  The Poultry Science department at Penn State University was consulted via their Agricultural Extension Service. From this interchange came a critical observation – the affected hens laid eggs that had white whites! Normal chicken egg white is yellow (see right; photographs from our University of Delaware colleague Dr. Hal White).

The yellow color of normal egg white reflects its content of vitamin B2 (aka riboflavin) to provide the growing embryo with this essential water-soluble vitamin. So the Penn State researchers injected the eggs with riboflavin and, mirabile dictu, normal-looking chicks emerged after 3-weeks incubation.

These mutant birds are unable to efficiently load riboflavin into their eggs because they fail to make the riboflavin-binding protein needed to transport the vitamin. A structure of riboflavin-binding protein loaded with riboflavin is shown here.

It was Hal White who made the key observation that led to the discovery of the Quiescin-sulfhydryl oxidase (QSOX) family of disulfide-generating enzymes. While the mutant egg whites were severely deficient in riboflavin, Hal found that they contain small amounts of a different form of flavin (flavin adenine dinucleotide, FAD) – a derivative of the vitamin one expects to be associated with redox enzymes. Since no FAD-linked enzyme had been described in egg white the Thorpe and White labs jointly sought to isolate and identify it – just out of curiosity. This project started modestly, but eventually required cracking many thousands of mutant eggs and purifying the FAD-binding protein from hundreds of liters of egg white. For these heroics we are most grateful to Dr. Karen Hoober who performed many of the critical early experiments while a graduate student at Delaware.  Our joint work led to the discovery that the FAD-binding protein was a disulfide-generating enzyme capable of facile catalysis of the reaction shown here.

Sequencing peptides from the egg white enzyme led to the realization that it shared an evolutionary kinship with a human growth factor Quiescin Q6 (discovered by Dr. Donald Coppock and colleagues). We jointly named these enzymes Quiescin-sulfhydryl oxidases – abbreviated QSOX because other obvious abbreviations hade been taken! QSOXs are found in most non-fungal eukaryotes – from diminutive marine algae to humans. Now the challenge is to learn their roles within cells and when they are deployed extracellularly.

QSOXs were independently discovered by Benayoun et al. – again serendipitously. They initially thought they had found a new cysteine protease inhibitor in seminal vesicle fluid … but it was a sulfhydryl oxidase that oxidized the dithiothreitol used as a protective agent in their assays! When they sequenced protein fragments from this “protease inhibitor” they also made the connection with Quiescin Q6. They had stumbled across the same yellow enzyme studied by Chang and Morton and by Ostrowski and Kistler more than 25 years earlier.

Acknowledgments, References and etc.

We are most grateful to the poultry extension specialist, Mr. Leuschner, at Penn State who observed those white whites almost 60 years ago! At the University of Delaware, Hal White and Colin Thorpe thank the Department of Animal and Food Sciences and Mr. Robert Alphin for their support over the years. Colin Thorpe particularly wishes to acknowledge Drs. Karen Hoober, Joan Burnside, Donald Coppock and Hal White for their contributions to the discovery of the QSOX family in Delaware.

Our 1996 paper on the isolation and characterization of sulfhydryl oxidase from egg white is Hoober et al.

The discovery of Quiescin Q6 by Coppock and colleagues is here [PubMed]

An abstract of an early report (1954) from Dr. Maw of Penn State is here.

More information about these eggs and the insights that led from them can be found in a multipart demonstration/presentation made by Hal White to a group of Delaware students – you could start with part 2:

The link to Hal White’s website is here and an excellent problem-based learning handout including more details of the discovery of the mutation in chickens is here

A list of QSOX-related developments can be found in the QSOXology section of this website. A recent review [PubMed] provides a reasonably current picture of the QSOX field (as of 2010). A complete list of our papers is elsewhere on this site.

The crystal structure coordinates of riboflavin binding protein were graciously supplied by Dr. Hugo L. Monaco. His paper describing the structure of RBP is here. [PubMed]

The article describing the Quiescin Q6 sulfhydryl oxidase link in seminal vesicles is here [PubMed]