Philip Petra
Emeritus Professor of Biochemistry

 

CURRICULUM VITAE

PHILIP HUE PETRA

(Revised – 2000, 2010, 2023, 2025)

Personal Data

Birthday: May 12, 1937
Birthplace: Paris, France
Citizenship: U.S., 1962
Marital Status: Married, Dolores
Children: Philippe
Anita

Education

B.S. Department of Chemistry
Tulane University
New Orleans, LA		 1955-60
M.S. Department of Biochemistry
Tulane University
New Orleans, LA
Dr. Elliot Shaw, thesis advisor
Thesis: A kinetic analysis of the catalyzed hydrolysis of α-N-benzoyl-l-citrulline methyl ester by papain		 1960-62
Ph.D. Department of Biochemistry
Tulane University
New Orleans, LA
Dr Gunther Schoellman and Dr. Elliott N. Shaw, thesis advisors
Thesis: Isolation and characterization of the alkylated histidine residue from trypsin inhibited by a specific reagent, TLCK		 1962-66
Postdoctoral Research Professor Hans Neurath
Department of Biochemistry
University of Washington Seattle, WA
Research: Structure-function relationship of Carboxypeptidase A		 1966-70

Academic Appointments

Position Departments Institution Dates
Acting Assistant Professor Department of Biochemistry University of Washington, Seattle, WA 1968-70
Assistant Professor Department of Obstetrics/Gynecology (joint with Biochemistry 1970-74; adjunct with Biochemistry 1974-75) University of Washington, Seattle, WA 1970-75
Assistant Professor with tenure Departments of Ob/Gyn and Biochemistry University of Washington, Seattle, WA 1975-78
Associate Professor Departments of Ob/Gyn and Biochemistry University of Washington, Seattle, WA 1978-84
Professor Departments of Ob/Gyn and Biochemistry University of Washington, Seattle, WA 1984-93
Professor Department of Biochemistry University of Washington, Seattle, WA 1993-2006
Professor Emeritus of Biochemistry – University of Washington, Seattle, WA 2006 –

Societies

  • The American Society for Biochemistry and Molecular Biology
  • The Protein Society
  • The Endocrine Society
  • The American Association for the Advancement of Science

Honors and Invitations

  • Exchange Scientist of the National Cancer Institute with Institut National de la Sante et Recherche Medicale, Paris, France, 1977.
  • Speaker, International Congress on Hormonal Steroids, New Delhi, India, 1978.
  • Speaker, International Congress on Hormonal Steroids, Jerusalem, Israel, 1982.
  • Symposium and speaker, Society for Gynecological Investigation, Washington, D.C., 1983.
  • Speaker, 7th International Symposium of the Journal of Steroid Biochemistry, Seefeld, Austria, 1985.
  • Visiting Professor with Dr. Etienne-Emile Baulieu, Institut National de la Sante et de la Recherche Medicale (INSERM) Paris, France, Sept 1985 – Sept 1986.
  • Seminar speaker, University of Geneva, Geneva, Switzerland, February 1986.
  • Seminar speaker, CNRS, Caen, France, March 1986.
  • Scientific Organizing Committee and speaker, First International Symposium on Steroid Hormone Binding Proteins, Lyon, France, April 1986.
  • Seminar speaker, INSERM U34, Lyon, France, May 1986.
  • Scientific Organizing Committee and speaker, 2nd International Congress on Steroid-Protein Interaction, Torino, Italy, September 1987.
  • Scientific Organizing Committee and speaker, 3rd International Congress on Steroid Binding Proteins, The Hague, The Netherlands, September 1990.

Teaching Responsibilities

1. Training responsibilities on NIH Ob/Gyn Departmental Training Grant. 1971 – 1978
2. Biochemistry 406 – General principles of Biochemistry
Class size – 250-500		 1978 – 2006
3. Biochemistry 426 – Basic Techniques in Biochemistry.
Class size – 40 students.		 1994 – 2000
4. Biochemistry 499 – Undergraduate Research. 1978 – 2000
5. Hu-BIO 514 1996 – 1997
6. Biochemistry 540 – Literature Review for biochemistry graduate students.
7. Biochemistry 581 – Laboratory rotation for biochemistry graduate students.

Research Funding: Principal Investigator

1. Graduate School Research Fund (Univ. of Wash.)
Title: Steroid Binding Proteins.
December 1971 – December 1972		 $2,935
2. American Cancer Society Institutional Grant.
Title: Biochemical studies on estradiol induction of metabolism in the uterus.
February 1973 – January 1974		 $2,400
3. NIH. The National Institute of Child Health and Human Development.
Title: Structure-Function of Steroid-binding Proteins
May 1972 – April 1975		 $94,400
4. American Cancer Society Institutional Grant.
Title: A filter assay for estrogen receptors in human mammary tumors.
December 1974 – December 1975.		 $1,200
5. NIH. The National Institute of Child Health and Human Development.
Title: Structure-Function of Steroid-binding Proteins.
June 1975 – May 1979.		 $124,114
6. American Cancer Society.
Title: Study of breast neoplasia with advanced receptor methodology.
July 1976 – July 1978.		 $71,085
7. Biomedical Research Support Grant (Univ. of Wash.).
Title: Influence of Steroid-binding plasma proteins on the growth of estrogen-dependent breast cancer.
July 1978 – March 1979.		 $4,356
8. NIH. The National Institute of Child Health and Human Development.
Title: Characterization of the plasma sex steroid-binding protein.
April 1981 – March 1988.		 $568,243
9. NIH. The National Institute of Child Health and Human Development
Title: Characterization of the plasma sex steroid-binding protein.
April 1988 – March 1993.		 $620,000
10. ROYALTY RESEARCH FUNDS, University of Washington
Title: Cloning of the Sex Steroid-Binding Protein.
January 1994 – May 1995.		 $34,230
11. NIH. The National Institute of Child Health and Human Development.
Title: Structure of plasma sex steroid-binding protein.
April 1995 – May 2001.		 $400,000

 

Research Activity

My interest in structure-function relationships in proteins began during my graduate work at Tulane University in 1960 under the guidance of Drs Elliott Shaw and Gunther Schoellman who had originated the method of affinity labelling of proteins. That chemical approach, designed to understand enzymology and protein function, consists of synthesizing small reactive molecules resembling enzyme substrates that would react with amino acid side chains in active sites thus identifying possible catalytic or other functional groups. With this approach, they were able to covalently link the label to a histidine residue in the active site of chymotrypsin, later identified as His-47 that functions in the hydrolytic action of the enzyme. At the time few sequences of proteins were known (insulin, ribonuclease, and lysozyme) and that of both chymotrypsin and trypsin had not yet been determined. For my PhD thesis, I was given the task of characterizing the chemical attack of the analogous histidine residue in trypsin by the affinity label, L-1-chloro3-tosylamido-7-amino-2-heptanone (TLCK), a chloro-methyl ketone which Shaw and co-workers had shown to inhibit trypsin in a 1:1 mole ratio. I accomplished this task and found that the reactive group of the affinity label had covalently bound to nitrogen-3 of the imidazole ring of a histidine residue (1), later identified as His-57 when Walsh et al. solved the amino acid sequence of trypsin. Another project on the kinetic analysis of papain-catalyzed hydrolysis was also completed (2).

After completing my theses, I moved to Seattle as a postdoctoral fellow with Professor Hans Neurath at the University of Washington. My first task was to solve the purification of bovine carboxypeptidase (CP-A) which had been elusive (3,5). I then turned my attention to the active center of the enzyme, the amino acid sequence of which had now been solved by Bradshaw, Walsh, and Neurath. The 3-D structure, solved by Lipscomb at Harvard, revealed the presence of two glutamic acid residues in the active center (Glu-72 and Glu-270), one possibly serving as catalytic group. I was given the task of identifying that residue chemically. I chose N-ethyl-5-phenylisoxazolium-3’-sulfonate (reagent K) as a carboxyl group-specific reagent because this reagent incorporates some of the structural features of small substrates of CP-A and thus could serve as an affinity label. R. B. Woodward (for whom reagent K is named) had used it for organic synthesis, and the Tulane Shaw group had used a similar reagent for the modification of carboxyl groups in proteins. Reagent K was found to inhibit CP-A in the absence of competitive inhibitors but not in their presence (6). The label attached to a carboxyl group in the enzyme active site as a mixed anhydride. I chose to stabilize it by nucleophilic displacement with 14C-methoxamine converting the attached label to a stable radioactive amide linkage. The labelled residue was identified as Glu-270 by purifying and sequencing radioactive peptides (7). The data identified Glu-270 as the catalytic group in enzymatic hydrolysis.

After completing other projects in the Neurath group (4,8), I started my faculty appointment at the University of Washington in 1970. I was offered an assistant professorship by Walter Hermann, chairman of the department of Ob/Gyn, and by Hans Neurath, chairman of the department of Biochemistry. The appointment was joint with teaching responsibilities in the Biochemistry department, and research responsibilities in the Ob/Gyn department. I directed my attention to steroid-binding proteins which had been implicated in steroid hormone action. These are present in plasma and target cells and specifically bind sex steroids with high affinity, such as dihydrotestosterone (DHT), testosterone (T), and estradiol (E2). Dr. Hermann et al. had discovered an estrogen receptor in brain tissue, so there was already experience in this field in the Ob/Gyn department. At the time none of these proteins had been characterized, sequenced, or cloned. The major problem for protein characterization was their low concentrations in both cells and plasma. I decided on the human plasma protein called Sex Steroid Binding protein (SBP) discovered by Etienne Baulieu’s group in the 1960s (9). This protein specifically binds DHT, T, and E2 with high affinity. The acronym SBP was given by Baulieu’s group, and it was adopted at the IVth Meeting of the International Study Group for Steroid Hormones, Rome, 1969. Unfortunately, other laboratories named the protein with different acronyms, such as SHBG (Sex Hormone Binding Globulin), TeBG (Testosterone estradiol Binding Globulin), and others, which caused some confusion in the field to this day.

I wrote my first NIH grant in 1972 and secured funding for SBP research. The first goal was to purify and characterize the protein as a model for studying structure-function relationships in steroid binding proteins. The plasma concentration SBP increases significantly during human pregnancy, so we obtained serum left over in the Ob/Gyn delivery rooms as a source of protein. The low plasma levels in that source required development of an efficient purification method. First, a DHT derivative, 3-oxo-17β-hydroxy-5α-androstane-17α-6-hexanoic acid was synthesized with the help of Niels Anderson of the Chemistry Department at the University of Washington. This was coupled with diamino-ethyloxirane-agarose to yield 5α-dihydrotestosterone-17α-hexanyldiaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, the affinity adsorbent for SBP purification (23). Non-human primate plasma was obtained from the Oregon Primate Center in collaboration with Miles Novy and Frank Stanczyk. These and other sources led to the purification and characterization of hSBP (11, 17), nSBP (macaque nemestrina SBP) (31), rhSBP (macaque mulatta SBP) (31), bSBP (baboon SBP) (40), and rSBP (rabbit SBP) (18). The amino acid sequence of hSBP was determined by subtractive Edman degradation (42). The sequence contains 373 residues, three carbohydrate chains one O-linked to Th-7 and the other two N-linked to Asn-351 and Asn-367, and two disulfide bonds Cys164-Cys188 and Cys333-Cys36. A carbohydrate content of 14% was determined by standard methods (31). The minimum molecular weight of hSBP is 47,000 as calculated from the amino acid sequence including the carbohydrate content (42). A lower value of 36,335 was obtained by equilibrium sedimentation in 6 M guanidine.HCl (17), and a higher value of 52,000 with SDS-Gel electrophoresis (17). The lower value may be explained by errors in the specific volume of the protein in the presence of denaturant, while the higher value comes from the presence carbohydrate which tends to yield abnormally high molecular weights in SDS-PAGE and gel filtration for glycoproteins (71,72,73). The amino acid sequence of rSBP determined by mass spectrometry contains 367 residues, with only two N-linked carbohydrate chains at Asn-345 and Asn-361, and two disulfide bonds connecting Cys-158 to Cys-182 and Cys-327 to Cys-355 (50). The molecular weight of rSBP calculated from the sequence including 9% carbohydrate is 43,702. The rSBP amino acid sequence is 79% identical to that of hSBP indicating that the two proteins are homologous and have arisen from a common ancestor; they comprise a gene family which includes rat androgen-binding protein ABP (76). Characterization of nSBP indicates a very close similarity to hSBP thus making it a good animal model for studying the physiological role of SBP (14). All three proteins have essentially the same Kd for DHT binding (0.3 nM to 0.9 nM at 4oC). Interestingly, the SBP/ABP gene family is distinct from the nuclear steroid hormone receptor superfamily because the cDNA-deduced sequences of the steroid binding domain of the human androgen receptor (74, 75) and estrogen receptor (70) are not homologous to the SBP sequence, although they all bind DHT with similar Kds (~ 0.2 nM at 4oC). The structural basis of the two different protein designs that lead to similar steroid-binding specificity is certainly an interesting research undertaking.

The molecular weight of native hSBP has been a contention issue since its discovery. We and others had reported values ranging from 88,000 to 120,000 as determined by gel filtration (17, 79). As noted above, gel filtration yields inaccurate values for glycoproteins which are more hydrated than polypeptide proteins tending them to be excluded from gel beads; this leads to an earlier elution in column chromatography and over-estimated values of molecular weight. In the case of SBP, the issue was resolved by equilibrium sedimentation under native conditions in the presence of saturating concentration of steroid (DHT). That method yields native molecular weights of 85,600 ± 3,000 for hSBP and 85,800 ± 4,000 for rSBP (40). These experimentally determined values are very close to those calculated from the amino acid sequences of each protein including the carbohydrate content, 94,000 for hSBP and 87,404 for rSBP. The data represents the first evidence that not only is SBP a dimer, but also a homodimer. The conclusion was rigorously supported by showing that there are only four unique sequences around the half-cystine residues in the amino acid sequences of each protein (38, 50). If SBP were a heterodimer, eight unique sequences instead of four would have been found around those residues. The steroid binding stoichiometry was obtained by steady-state electrophoresis of pure SBP in the presence of [H3]DHT (14, 31). Steady state means that the label is included during polymerization of the gel prior to electrophoresis to ensure that the label stays bound to SBP during electrophoresis (16). Labeled SBP was extracted from cut-out gel slices and counted, the molar SBP concentration was obtained spectrophotometrically at 280 nm using the molar extinction coefficient, ε280, 1.14 x 105 cm-1 M-1 (that value was determined from at least 6 different pure hSBP preparations). The steroid-bound concentration was calculated using the specific radioactivity of the label (Ci/mmole). The results showed a steroid binding stoichiometry ranging from 0.93 to 1.32 mole bound ligand per mole dimeric SBP (31, 40). Variation is due to small amounts of inactive SBP molecules that may be present in the mixture. It should be noted that determination of stoichiometry by this method requires that affinity chromatography be included as one of the steps in SBP purification to ensure that the isolated purified SBP is active. In conclusion, the data shows that native SBP binds DHT with a 1:1 mole DHT/mole dimer.

A one-to-one binding stoichiometry of a ligand to a homodimeric protein presents an interesting structural problem. We originally proposed that the steroid was bound at the interface between the subunits to account for the specific recognition of an asymmetric ligand, such as the steroid, by a homodimer SBP (60, 79). In this way a different face of each monomer would recognize the asymmetric ligand. This proposal was supported by the fact that the best affinity adsorbent for SBP purification must contain positions C-17β of ring D and C-3 of ring A of the steroid both exposed; if one is blocked by the spacer linked to the matrix, the purification yield is dramatically reduced (11, 17, 23). This means that the steroid can insert into the site from either end; so, placing the site at the interface between the subunits would provide easy access. However, we also proposed another model for explaining the 1:1 stoichiometry, one in which the binding of the first steroid to one monomer induces an allosteric conformation in the other resulting in inhibition of binding in the second site (79). Positive and negative cooperativity in the function of multimeric proteins had been proposed by Monod, Changeux, Wyman, and Koshland in the 1960s, they provided two models for explaining allostery. For example, resolution of hemoglobin structure revealed the various allosteric structural steps involved in oxygen binding. In this context, we found that some SBP preparations kept frozen for extended periods appeared to bind more than one mole steroid per dimer (78) suggesting an irreversible change in conformation abolishing negative cooperativity of binding.

While structural studies were advancing, research on the physiological role of SBP was initiated. Availability of pure hSBP, nSBP, and monospecific hSBP antibodies (19), as well as collaborations with M. Novy and F. Stanczyk at the Oregon Primate Center, and S. R. Plymate at Madigan Army Medical Center in Tacoma WA, allowed research on non-human primates. Prior to the discovery of SBP, clinical studies in the 1960s had shown that unbound steroids in human plasma diffused nonspecifically into tissues. This idea was supported by measuring the metabolic clearance rate (MCR) of sex steroids in males and patients with low and high SBP levels. Faster MCR-T was found in males with low SBP values, and slower MCR-T was found in pregnant females who have high levels of SBP. With the discovery of SBP, it was proposed that the protein functions by regulating the diffusion of sex steroids into tissues by a steady-state equilibrium in plasma. With access to pure protein and its antibodies we could explore those questions. Infusion of pure nSBP and purified anti-hSBP which cross-react with macaque nemestrina SBP (19) into the macaque nemestrina provided rigorous proof that SBP has a direct effect on MCR-T and MCR-E2. MCR-T decreased following the infusion of nSBP and increased following the infusion of hSBP antibodies (33, 51). Furthermore, incubation of endometrial cancer cells in culture with pure hSBP inhibited the intracellular diffusion of E2 (35). These results support the premise that the unbound (“free”) steroid fraction in plasma is the active form of the hormone that enters cells and tissues. The “free” T concentration in plasma is calculated from the Kd of T bound to albumin (5 x 10-5 M at 37oC), the Kd of T bound to SBP (4 x 10-9 M at 37oC), and the plasma concentrations of total T, albumin, and SBP (16). There lies the role of SBP, it controls the level of “free” steroid in plasma by a steady-state equilibrium between SBP and albumin. Plasma albumin is absolutely required for this regulation. Another role of SBP has been suggested by us (25) and others, which involves the possible existence of an SBP receptor acting in active transport of steroids into cells. Such a receptor was never found and currently there is insufficient data to support such a proposal.

It became clear that continuing research on SBP would require training in molecular biology and cloning methods. I therefore took a sabbatical year’s leave of absence to learn these techniques. Dr. Etienne Baulieu invited me as a visiting professor at INSERM Unit 33, Paris, in 1985. On my return to Seattle, I secured a collaboration with Dr. Fred Hagen of Zymogenetics at the time and began the cloning and expression of hSBP-cDNA. We did not isolate a full-length clone (46) and decided to construct it from smaller fragments (54). This cDNA was expressed in mammalian cells (54, 56), insect cells (58), yeast (64), all yielded active recombinant SBP. Site-directed mutagenesis revealed Tyr-57 (66), Met-107 (66), Lys-134 (53, 56), and Met-139 (56) as functional in steroid binding. Two of these residues (Tyr-57 and Lys-134) are at homologous positions in rSBP (Tyr-51 and Lys-128) but Met-107 is replaced with an isoleucine (Ileu-101) in rSBP. Since rSBP binds E2 ten-fold less than androgens, we initiated mutagenesis experiments to identify the residues responsible for E2 binding in hSBP. The difference in the 3-D structures of DHT and E2 is small and located in the A-ring of the steroids, so we expected that replacement of not more than two residues would be sufficient to obtain a loss in E2 binding of hSBP. Fifty-six replacements were found by aligning both the human and rabbit sequences starting at the amino terminus. Combinatorial oligonucleotides coding for the changes were synthesized for each selected segment. Their expression shows that all of them had wild type DHT Kd values (~ 0.4nM) and all had wild type E2 Kd values (~ 4 nM), except those having the R140K and I141L replacements which had E2 Kds of ~ 40nM, close to that of the rSBP E2 Kd (~ 80 nM). So, it appears that R-140 and I-141 determine E2 specificity in hSBP and that these residues must be in or near the steroid-binding site of hSBP where the A-ring of E2 is recognized (67).

In 2000 I entered a collaboration with Dr. Wim Hol to crystallize the native and recombinant proteins for initiating resolution of the 3-D structure of hSBP. We had previously expressed both wild-type and fully-deglycosylated protein in yeast (64). In the meantime, a group in Germany published a crystal structure of the N-terminal domain of hSBP (residues 1 to 205) representing about 60% of the sequence (80). This fragment crystallized as a dimer with two steroid binding sites, suggesting a 2:1 binding stoichiometry for native dimeric SBP. No mention was made on the possibility that the missing C-terminal portion (40% of the sequence) could function in negative cooperativity by blocking the second site from binding the ligand thus explaining the 1:1 stoichiometry that we and others had proposed. Although the Muller model clearly showed that the steroid binding site was not located at the interface between the subunits, as we had predicted, I became convinced that SBP must be under allosteric control in which one binding site is functional under normal DHT concentration and normal SBP concentration (~ 20 nM in human plasma) while the other is repressed under those conditions. As demonstrated in the Muller model, the two binding sites are located in the amino terminal regions of each subunit which is consistent with our affinity labeling and site directed mutagenesis data (53, 56, 66, 67). Lys-134 could not be identified due to lack of X-ray data between residues 130 and 135. Recall that hSBP and rSBP have similar DHT Kds (0.42nM and 0.86 nM at 4oC, respectively) but very different E2 Kds (4.79 nM and 84.6 nM at 4oC, respectively). Moreover, the human to rabbit replacements R140K and I141L yielded mutants having E2 Kds of about 40nM, close to that of the rSBP E2 Kd, suggesting that R-140 and I-141 might function in determining E2 specificity of hSBP. So, in collaboration with Dr. Elinor Aldman, department of Biological Structure at UW, we modeled the triple mutant M107I/R140K/I141L into the Mueller model to explore the structural basis of estrogen binding specificity in hSBP (the M107I replacement has no effect on DHT or E2 binding (67)). No structural changes between wild type hSBP and the triple mutant were found when DHT was modeled into the site. However, when modeling E2 into the site, there were significant structural changes that allowed the phenyl ring of F56 to move closer to ring A of 17β-estradiol thus optimizing E2 binding affinity to hSBP. The side chains of R140 and I141 in the wild-type Muller model do not contact the steroid, precluding direct interaction as the explanation for the differences in E2 affinity. Instead, the replacements disrupted the network of H-bonds that stabilized the packing of ring A of E2 with F56. Interestingly, the structural change does not occur within the active site but at some distance where R140 and I141 are located. Such a finding suggests a strategy in the molecular evolution of SBP that allows the gradual addition of a new function without destroying the existing beneficial one. It appears that the ability of hSBP to bind E2 was superimposed onto the original androgen binding design and probably occurred more recently in evolutionary times. Moreover, the R140K and I141L replacements in the molecular evolution allow hSBP to extend its function to the female of the species where regulation of “free” estradiol concentration plays a fundamental hormonal role.

Additional comments added in Spring 2025

To remain current on recent development in the SBP field, I decided to collaborate with Christophe Verlinde (Professor emeritus, UW Department of Biochemistry) to review the literature on SBP structure during the past 25 years since I closed my laboratory. The survey indicates that the preferred acronym by clinical endocrinologists is SHBG. To my amazement we found only seven significant papers on aspects of SBP structure published by two different laboratories. As discussed above, by the time I retired the Muller group had published a crystal structure of the N-terminal domain representing about 60% of the amino acid sequence of human SBP (80). They had shown that the steroid binding site was located entirely in that fragment and because it crystallized as a dimer, they concluded that SBP binds two moles steroid per mole dimer. They have now published five additional papers expanding on SBP structure. New evidence for the location of residues 130 to 135 in the steroid binding site is presented (81); that segment contains Leu-131 and Lys-134 we had previously identified in the binding site (53). In another paper they reveal that estradiol occupies the SBP site in the opposite direction to that of DHT (82). We predicted this interesting result years ago when designing affinity adsorbent for SBP purification by showing that sex steroids containing positions C-17β of D-ring and C-3 of A-ring left exposed yield the highest amount of pure protein; if either one is blocked by the spacer linking it to the gel matrix, the purification yield is dramatically reduced (11, 17, 23). This means that the steroid binding site is “open” at both ends allowing the ligand to enter the site in either direction. Although we modeled estradiol and DHT in the same orientation, in collaboration with Dr Elinor Aldman (67), we had no reason to expect significant differences by modeling them in opposite direction. However, we agree with the authors that the use of homologous modeling is a poor substitute for experimentally determined crystal structures. Another paper analyzes the structural properties of C-2 derivatives of estradiol bound to SBP (83).

Their last paper (84) requires critical discussion. The Muller group previously identified a dimerization domain between the truncated SBP monomers (lacking the C-terminal domain). In this paper they mutate some of these residues to explore their possible role in dimerization. The mutants were made in the entire SBP amino acid sequence containing the C-terminal domain. They claim to have generated active steroid binding monomers and conclude that native SBP binds 2 moles steroid per mole dimer and that binding of sex steroids by SBP has been underestimated by a factor of 2. However, the data presented in that paper is not sufficiently rigorous to support such a conclusion. First, no physiochemical characterization of the three recombinant SBPs containing mutations was carried out to ensure accurate determination of molecular weight of the V89E and other mutants. Only gel filtrations are carried out which is known to lead to erroneous values for glycoproteins (72, 73, 74). V89E elutes as a broad radioactive peak with molecular weights ranging from 80,000 to 50,000, close to that of native dimeric SBP when purified by the same method (17, 79). Recall that monomeric SBP molecular weight is 47,000 as determined from the sequence (42) and 36,335 by sedimentation equilibrium (17). Most of the broad radioactive peak of V89E elutes outside this range. Second, because the V89E mutant was not purified and characterized, one cannot be certain that the broad radioactive peak of V89E represents a monomeric form of SBP or denatured forms of dimeric SBP caused by the mutation. Recall that SBP monomers are not found in human plasma, likely because they are unstable and can stabilize only by forming dimers. In native SBP, it is dimerization that generates the two potential steroid binding sites, one functional under physiological conditions and the other repressed through allosteric modulation. Thirdly, they report a DHT Kd for V89E of 1.1 nM which is approximately ten times higher than that of wild-type SBP, 0.3nM at 40C (14, 18). Moreover, the binding data was not shown as Scatchard plots to assess both accuracy and stoichiometry, they certainly had enough data to calculate at least an approximate binding stoichiometry for V89E. In conclusion, V89E and the other two mutants are not likely to represent recombinant monomeric SBP and one cannot say from such data that monomeric SBP binds DHT. The mutants are probably forms of denatured or unfolded dimeric SBPs that still bind DHT but with less affinity than the native dimer. To be noted, a negative cooperativity allosteric effect proposed by us and others for explaining 1:1 stoichiometry is not mentioned anywhere in that paper. We think it is appropriate here to suggest that, when doing science, one should always use rigorous methodologies especially when challenging existing paradigms (85).

Lastly, we review two interesting papers published by a new group in the field, Jasuja and coworkers. The first paper (86) takes up the clinical issue on how to estimate levels of free T in plasma in the diagnosis and treatment of hypogonadism in men. The Endocrine Society has suggested the use of a calculated value for estimating free T. In this paper they show that the Muller model showing a homodimer having two steroid binding sites with the same binding affinity regardless of ligand occupancy is incorrect. Calculating free T using that model does not fit their steroid binding isotherms. Even modeling a simple negative cooperativity, as we had proposed, does not fit the experimental data. Instead, they describe a new model which depicts a multi-step dynamic process in which, in the absence of T, the dimeric SBP molecule exists in at least two inter-converting conformations in dynamic equilibria. Binding T to the first monomer affects the interaction of T with the unoccupied second binding site of the second monomer. As T concentration increases there is a dynamic re-adjustment in the structure of intermediates in such a way that allows SBP to regulate free T levels in a much larger dynamic range of T concentration. This complex model, model G depicted in Fig 3 of their paper, fits the experimental data obtained by equilibrium dialysis varying either T or SBP concentrations, as well as T depletion and isothermal titration calorimetry curves. This finding unveils an unexpected structural characteristic of the SBP dimeric molecule, which had eluded us in our original work, endowing it with functions beneficial for regulating steroid binding at a wider range of T and SBP concentrations. SBP appears to function like a “buffer” maintaining a constant steady-state free T concentration not only in normal physiological conditions but also in disease or pharmacological situations where T levels may rise abnormally. The function is analogous to a buffer maintaining a constant pH during an increase of acidity. The resulting negative cooperativity is expressed through this complex allosteric mechanism. The molecular evolution of this phenomenon is remarkable, instead of creating a new gene, the existing one encoding the monomer is slightly changed to include a dimerization domain allowing formation of a homodimer. This genetic modification gives rise to a new function by creating an allosteric regulation that blocks the second site from being occupied at normal physiological concentration of the ligand; however, the second site is allowed to function when [T] rises abnormally. It seems that the SBP structure is endowed with the ability to extend its function and protect the organism by maintaining constant “free” [T] even when total [T] rises abnormally.

In their second paper the authors extend their work to E2 binding to SBP (87). As in the case of T, E2 binding to hSBP shows nonlinear isotherms as performed by equilibrium dialyses in a wide range of E2 concentrations. Dialysis shows nonlinearity in E2 Kd as the hSBP concentrations and hSBP/E2 ratios changed. That variation indicates multiple equilibria in the E2-SBP interaction with distinct affinities supporting their T work and the notion that the two monomers do not have equivalent E2 binding affinities. Using these approaches with molecular dynamics and Markov state modeling they conclude that there is an allosteric interaction between the two monomers upon E2 binding. The net effect is reshaping the free energy landscape of each monomer and stabilizing distinct conformational states. The data does not conform with the existence of two rigid and identical binding sites, instead it appears that E2 preferentially binds to the first monomer, but it is the second monomer that preferentially releases the steroid because the first monomer, in the fully bound state, reverts to a lower E2 Kd. The “free” ligand concentration dependent allostery makes physiological sense because it provides a molecular mechanism for maintaining a constant “free” E2 level in plasma when total E2 concentrations varies. We had previously shown that infusion of pure SBP and its antibodies controls the metabolic clearance rate of both testosterone and estradiol in the nonhuman primate (33, 51). According to these recent findings, the magnitude of sex steroid clearance from human plasma into target tissues (or degradation into liver) depends on both SBP and steroid concentrations, and those depend on the extent of negative cooperativity of binding to SBP.

Although these later findings are significant, we now need structural data to support the complex allostery proposed by the Jasuja group. The existing X-ray diffraction data was valuable to describe the location and structure of the steroid binding site of SBP, but that model cannot say anything about allostery because it lacks the C-terminal domain which is likely involved in the allosteric regulation. We repeatedly attempted to crystallize the native and full-length deglycosylated recombinant protein without success. However, we were able to image the dimeric SBP molecule by electron microscopy and most of the particles appear as short rods with a mean length of 19 + 3 nm and a thickness of 3 + 1 nm (61). Now the method of cryo electron microscopy which has greatly evolved in recent years should be attempted. In 1999 and 2000 I realized that continuing efforts would not likely lead to the production of crystals, much less diffractable crystals. Having completed most of our research objectives I decided to close the lab in 2001. I kept my teaching responsibilities until 2006 at which time I fully retired. In closing, I want to acknowledge Hans Neurath and Walter Herrmann for giving me the opportunity of coming to Seattle, joining the faculty of the University of Washington, and contributing to its research mission. In addition, I want to thank Ken Walsh, David C. Teller, and Elinor Adman for their contribution in understanding structure function relationships, Fred Hagen for his involvement in the molecular biology studies, and all the students and postdocs that participated in this research, they have been acknowledged in the publications listed below.

Bibliography

  1. Petra P.H., Cohen W., Shaw E.N.: Isolation and characterization of the alkylated histidine from TLCK-inhibited trypsin. Biochem. Biophys. Res. Comm. 21, 612-618, 1965.
  2. Cohen W., and Petra P.H.: A kinetic analysis of the papain-catalyzed hydrolysis of a-N-benzoyl-l-citrulline methyl ester. Biochemistry 6, 1047-1053, 1967.
  3. Petra P.H., and Neurath H.: The heterogeneity of bovine carboxypeptidase A. I. The chromatographic purification of carboxypeptidase (Anson). Biochemistry 8, 2466-2475, 1969.
  4. Petra P.H., Bradshaw R.A., Walsh K.A., and Neurath H.: Identification of the allotype amino acid replacements of bovine carboxypeptidase A. Biochemistry 8, 2762-2768, 1969.
  5. Petra P.H., and Neurath H.: The heterogeneity of bovine carboxypeptidase A. II. The chromatographic purification of carboxypeptidase (Cox). Biochemistry 8, 5029-5036, 1969.
  6. Petra P.H.: The modification of carboxyl groups in bovine carboxypeptidase A. I. The inactivation of the enzyme by N-ethyl-5-phenylisoxazolium-3-sulfonate (Woodward’s Reagent K). Biochemistry 10, 3163-3170, 1971.
  7. Petra P.H., and Neurath H.: The modification of carboxyl groups in bovine carboxypeptidase A. II. Chemical identification of a functional glutamic acid residue and other reactive groups. Biochemistry 10, 3171-3177, 1971.
  8. Petra P.H., Hermodson M., Walsh K.A., and Neurath H.: Characterization of bovine carboxypeptidase A (Allan). Biochemistry 10, 4023-4025, 1971.
  9. Mercier, C., Alfsen, A., and Baulieu E.E.: A testosterone binding globulin. In Proceedings of the 2nd Symposium on Steroid Hormones. Ghent, Belgium (1965). Excerpta Med. Int. Congr. Ser. 101 (1966) p. 212.
  10. Mickelson K.E., and Petra P.H.: A filter assay for the Sex Steroid-binding Protein (SBP) of human serum. FEBS Lett. 44, 34-38, 1974.
  11. Mickelson K.E., and Petra P.H.: Purification Sex Steroid-binding Protein (SBP) from human serum. Biochemistry 14, 34-38, 1974
  12. Schiller H., and Petra P.H.: A filter assay for the corticosteroid-binding globulin (CBG) of human serum. J. Ster. Bioch. 7, 55-59, 1976.
  13. Gellert R.J., Lewis J., and Petra P.H.: Neonatal treatment with sex steroids. Relationship between the uterotropic response and the estrogen receptor in prepubertal rats. Endocrinology 100, 520-528, 1977.
  14. Petra P.H., and Schiller H.: Sex Steroid-binding Protein (SBP) in the plasma of the Macaca nemestrina. J. Ster. Bioch. 8, 655-661, 1977.
  15. Schiller H., Langley J.W., and Petra P.H.: Corticosteroid-binding protein in the plasma of the Macaca nemestrina. J. Ster. Bioch. 8, 647, 1977.
  16. Tabei T., Mickelson K.E., Neuhaus S., and Petra P.H.: Sex Steroid-binding protein (SBP) in dogs. J. Ster. Bioch. 9, 983-988, 1978.
  17. Mickelson K.E., Teller D.C., and Petra P.H. Characterization of the sex steroid binding protein of human pregnancy. Improvement in the purification procedure. Biochemistry 17, 1409-1415, 1978.
  18. Mickelson K.E., and Petra P.H.: Purification and physico-chemical characterization of the Sex Steroid-binding Protein of rabbit serum. Comparison with the human protein. J. Biol. Chem. 253, 5293-5298, 1978.
  19. Bordin S., Lewis J., and Petra P.H. : Monospecific antibody to the Sex Steroid-binding Protein (SBP) of human and rabbit serum: Cross-reactivity with other species. Bioch. Biophys. Res. Comm. 85, 391-401, 1978.
  20. Namkung P.C., Moe R.E., and Petra P.H.: Stability of estrogen receptors in frozen human breast tumor tissue. Cancer Res. 39, 1124-1125, 1979.
  21. Heinrichs W.L., Tabei T., Kuwubara Y., Burry K., Resko J., Petra P.H., Schiller H., and Namkung P.C.: Differentiation and regulation of peripheral androgen metabolism in rats and rhesus monkeys. Am. J. Obstet. Gynec. 135, 974-983, 1979.
  22. Raijfer J., Namkung P.C., and Petra P.H.: Ontogeny of androgen receptor in penis. Surgical Forum, Vol. XXX, Chapter SVII, 1979.
  23. Petra P.H., and Lewis J.: Modification in the purification of the sex steroid-binding protein of human serum by affinity chromatography. Anal. Bioch. 105, 165-169,1980.
  24. Burry K.A., Tabei T., Resko J., Petra P.H., and Heinrichs L.W.: Differentiation of sex steroid-binding protein in adult rhesus monkeys. Am. J. Obstet. Gyn. 136, 446-450, 1980.
  25. Bordin S., and Petra P.H.: Immunocytochemical localization of the plasma Sex Steroid-binding Protein (SBP) in tissues of the adult male monkey, Macaca nemestrina. Pro. Natl. Acad. Sci. (USA) 77, 5678-5682, 1980.
  26. Raijfer J., Namkung P.C., and Petra P.H.: Identification, partial characterization, and age-related changes of cytoplasmic androgen receptor in the rat penis. J. Steriod Bioch. 13, 1489-1492, 1980.
  27. Raijfer J., Namkung P.C., and Petra P.H.: The ontogeny of the cytoplasmic androgen receptor in the rat ventral prostate gland. Biol. Repro. 23, 518-521, 1980.
  28. Namkung P.C., and Petra P.H.: Measurement of progesterone receptors in human breast tumors. Comparison of various methods of analysis. J. Steroid Bioch. 14, 851-854, 1981.
  29. Bordin S., Torres R., and Petra P.H.: An enzyme immunoassay for the Sex steroid-binding Protein (SBP) of human plasma. J. Ster. Bioch. 17, 453-457, 1982.
  30. Ross J.B.A., Torres R., and Petra P.H.: Equilenin, a specific fluorescent probe for steroid-protein interactions in Sex Steroid-binding Protein (SBP). FEBS Letters 149, 240-244, 1982.
  31. Turner E., Ross J.B.A., Namkung P.C., and Petra P.H.: Purification and characterization of the sex steroid-binding protein from macaque serum. Comparison with the human protein. Biochemistry 23, 492-497, 1984.
  32. Stanczyk F.Z., Petra P.H., Senner J.W., and Novy M.J.: Effect of dexamethasone treatment on sex steroid-binding protein, corticosteroid-binding globulin, and steroid hormones in cycling rhesus macaques. Am. J. Obstet. Gyn. 151, 464-470, 1985.
  33. Petra P.H., Stanczyk F.Z., Namkung P.C., Fritz M.A., and Novy M.J.: Direct influence of the sex steroid-binding protein (SBP) of plasma on the metabolic clearance rate of testosterone. J. Steroid Bioch. 22, 739-746, 1985.
  34. David G.F., Koehler J.K., Brown J.A., Petra P.H., and Farr A.G.: Light and electron microscopic studies on the localization of the Sex Steroid-binding Protein (SBP) in rabbit spermatozoa. Biol. Reprod. 33, 503-514, 1985.
  35. De Ryck L., Ross J.B.A., Petra P.H., and Gurpide E.: Estradiol entry into endometrial cells in suspension. J. Ster. Bioch. 23, 145-152, 1985.
  36. Wortsman T., Frank S., Wehrenberg W.B., Petra P.H., and Murphy J.E.: Melanocyte-stimulating hormone immunoreactivity is a component of the neuroendocrine response to maximal stress (cardiac arrest). J. Clin. Endocr. Metab. 61, 355-360, 1985.
  37. Ross J.B.A., Contino P.B., Lulka M.F., and Petra P.H.: Observation and quantitation of metal binding sites in the Sex Steroid-binding Protein of human and rabbit sera using the luminescent probe, terbium. J. Prot. Chem. 4, 299-304, 1985.
  38. Petra P.H., Kumar S., Hayes R., Ericsson L.H., Titani K.: Molecular organization of the sex steroid-binding protein (SBP) of human plasma. J. Ster. Bioch. 24, 45-49, 1986.
  39. Orstan A., Lulka M.F., Eide B., Petra P.H., and Ross J.B.A.: Steroid-binding site of human and rabbit Sex Steroid-binding Protein of plasma: Fluorescence characterization with equilinin. Biochemistry 25, 2586-2692, 1986.
  40. Petra P.H., Namkung P.C., Senear D.F., McCrae D.A., Rousslang K.W., Teller D.C., and Ross J.B.A.: Molecular characterization of the sex steroid-binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque, and baboon proteins. J. Ster. Bioch. 25, 191-200, 1986.
  41. Stanczyk F.Z., Hess D.L., Namkung P.C., Senner J.W., Petra P.H., and Novy M.J.: Alterations in Sex Steroid-binding Protein (SBP), corticosteroid binding globulin (CBG), and steroid hormone concentrations during pregnancy in rhesus macaques. Biol. Reprod. 35, 126-132, 1986.
  42. Walsh K.A., Titani K., Takio K., Kumar S., Hayes R., and Petra P.H.: Amino acid sequence of the sex steroid-binding protein of human blood plasma. Biochemistry 25, 7584-7590, 1986.
  43. Petra, P.H., Titani, K., Walsh, K.A., Joseph, D.R., Hall, S.H., and French, F.S. Comparison of the amino acid sequence of the sex steroid-binding protein of human plasma (SBP) with that of the androgen-binding protein (ABP) of rat testis, in: Binding Proteins of Steroid Hormones (Forest, M.G., and Pugeat, M. eds), , Colloque/INSERM, Vol. 149, pp. 137-142, 1986, John Libbey, London/Paris.
  44. Stanczyk F.Z., Namkung P.C., Fritz M.A., Novy M.J., and Petra P.H. The influence of sex steroid-binding protein on the metabolic clearance rate of testosterone. In: Binding Proteins of Steroid Hormones (Forest, M.G., and Pugeat, M. eds), Colloque/INSERM, Vol. 149, pp. 555-563, 1986, John Libbey, London/Paris.
  45. Petra P.H.: Measurement of the sex steroid-binding protein of human plasma by an enzyme-linked immunosorbent assay, ELISA. in: Binding Proteins of Steroid Hormones (Forest, M.G., and Pugeat, M. eds). Colloque/INSERM, Vol. 149, pp. 215-220, 1986, John Libbey, London/Paris.
  46. Que B.G., and Petra P.H.: Characterization of a cDNA coding for sex steroid-binding protein of human plasma. FEBS Letters 219: 405-409, 1987.
  47. Mercier-Bodard C., Radanyi C., Roux C., Groyer M.T., Robel P., Dadoune J.P., Petra P.H., Jolly D.J., and Baulieu E.E. (1987) Cellular distribution and hormonal regulation of hSBP in human hepatoma cells. J. Steroid Biochem. 27:297-307.
  48. Petra P.H., Que B., Namkung P.C., Ross J.B.A., Charbonneau H., Walsh K.A., Griffin P.R., Shabanowitz J., and Hunt D.F.: Affinity labeling, molecular cloning, and comparative amino acid sequence analysis of sex steroid-binding protein of plasma. A multidisciplinary approach for understanding steroid-protein interaction and its physiological role. Annals N.Y. Acad. Sci., Vol. 538, 10-24, 1988.
  49. Namkung P.C., Stanczyk F.Z., Cook M.J, Novy M.J., and Petra P.H. (1989) Half-life of Plasma Sex Steroid-binding Protein (SBP) in the Primate. J. Ster. Bioch. 32: 675-680 1989.
  50. Griffin R., Kumar S., Shabanowitz J., Charbonneau H., Namkung P.C., Walsh K.A., Hunt D.F., and Petra P.H. The amino acid sequence of the sex steroid-binding protein of rabbit serum. J. Biol. Chem. 264, 19066-19075, 1989.
  51. Plymate S.R, Namkung P.C., Matej L.A., and Petra P.H. Direct effect of plasma sex steroid-binding protein (SBP or SHBG) on the metabolic clearance rate of 17b-estradiol in the primate. J. Ster. Bioch. 36: 311-317, 1990.
  52. Casali E., Petra P.H., and Ross J.B.A. Fluorescence Investigation of the Sex steroid Binding Protein of Rabbit Serum: Steroid Binding and Subunit Dissociation. Biochemistry 29: 9334-9343, 1990.
  53. Namkung P.C., Kumar S., Walsh K.A., and Petra P.H. Identification of lysine-134 in the steroid-binding site of the sex steroid-binding protein of human plasma. J. Biol. Chem. 265: 18345-18350, 1990.
  54. Hagen F., Arguelles C., Sui L.M., Seidel P., Conroy S.C., & Petra P.H. Mammalian Expression of the Human Sex Steroid-Binding Protein of Plasma (SBP or SHBG) and Testis (ABP). Characterization of the Recombinant Protein. FEBS letters 299: 23-27 (1992).
  55. Petra PH, Griffin PR, and Moore K. Complete Enzymatic Deglycosylation of Native Sex Steroid-Binding Protein (SBP or SHBG) of Human and Rabbit Plasma. Effect on the Steroid-Binding Activity. Protein Science 1:902-909, 1992.
  56. Sui L.M., Cheung A.W.C., Namkung P.C., & Petra P.H. Localization of the Steroid-Binding Site of the Sex Steroid-Binding Protein (SBP or SHBG) of Human Plasma by Site-Directed Mutagenesis. FEBS Letters 310:115-118, 1992.
  57. Verlinde, C. L. M. J., Merritt, E. A., van den Akker, F., Kim, H., Fei, I., Delboni., L. F., Mande. S. C., Safarty. S., Petra, P. H., & Hol. W. G. J. Protein Crystallography and Infectious Diseases. Protein Science 3:1670-1686, 1994.
  58. Sui L.M., Wong C., & Petra P.H. Over-expression of Human Sex Steroid-Binding Protein (hSBP/hABP or hSHBG) in Insect Cells Infected with a Recombinant Baculovirus. Characterization of the Recombinant Protein and Comparison to the Plasma Protein. J. Steroid Biochem. Molec. Biol. 52:173-179, 1995.
  59. Kim, H., Feil, I., Verlinde, C. L. M. J., Petra, P. H., & Hol, W. G. J. Crystal Structure of Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase from Leishmania mexicana: Implications fro Structure-Based Drug Design and a New Position for the Inorganic Phosphate Binding Site. Biochemistry 34:14975-14986, 1995.
  60. Sui L.M., Hughes W., Hoppe A.J. & Petra P.H. Direct Evidence for the localization of the Steroid-binding site of the Plasma Sex Steroid-binding Protein (SBP or SHBG) at the Interface between the Subunits. Protein Science 5:2514-2520, 1996.
  61. Beck K., Gruber T., Ridgway C. C., Hughes W., Sui L.-M., and Petra, P.H. Secondary Structure and Shape of Plasma Sex Steroid-Binding Protein (SBP or SHBG). Comparison with Domain G of Laminin Results in a Structural Model of SBP. Eur. J. Biochem. 247:339-347 (1997)
  62. Bernstein B. E., Michels P.A.M., Kim H., Petra P.H., Hol W.G.J. The importance of dynamic light scattering in obtaining multiple crystals forms of T. brucei PGK. Protein Science 7:504-507 (1998)
  63. Mankoff D.A., Tewson T. J., Gralow J.R., Petra P.H., Peterson L.M., Woo I., Yaziji H., and Gown A.M. [18F]-16α-fluoroestradiol (FES) and positron emission tomography (PET) to measure estrogen receptor expression in breast cancer. Breast Ca. Res. Treat. 50:332 (1998).
  64. Sui L.M., Lennon J., Ma C., McCann I., Woo I., & Pétra P.H. Heterologous expression of wild type and deglycosylated human plasma sex steroid-binding protein (SBP or SHBG) in the yeast, Pichia pastoris. Characterization of recombinant proteins. J. Steroid. Biochem. Mol. Biol. 68:119-127. (1999)
  65. Tewson T. J., Mankoff D. A., Peterson L. M., Woo I., and Petra P. H. Interaction of 16a-[F-18]-Fluoroestradiol (FES) with Sex steroid Binding Protein (SBP) Nuclear Medicine & Biology 26:905-913 (1999)
  66. Pétra P. H., Woodcock K., Orr W. R., Nguyen D., and Sui L. M. The sex steroid binding protein (SBP or SHBG) of human plasma: Identification of Tyr-57 and Met-107 in the steroid binding site. J. Steroid Biochem. Mol. Biol. 75:139-145 (2000)
  67. Petra P.H., Adman E.T., Orr W.R., Woodcock K.T., Groff C., and Sui L-M. Arginine-140 and Isoleucine-141 determine the 17b-estradiol-binding specificity of the sex steroid-binding protein (SBP or SHBG) of human plasma. Protein Science 10: 1811-1821 (2001).
  68. Mankoff D.A., Peterson L.M., Tewson T. J., Stekhova S.A., Petra P.H., Gown A.M., Gralow J.R., Livingston R.B., Schubert E.K., and Krohn K.A., Non-invasive PET imaging of ER expression in breast cancer: sex steroid binding protein (SBP or SHBG) interaction and ER expression heterogeneity. Proceedings of the AACR 42:6 (2001).
  69. Linden H.M., Stekhova S.A., Link J.M., Gralow J.R., Livingston R.B., Ellis G.K., Petra P.H., Peterson L.M., Schubert E.K., Dunnwald L.K., Krohn K.A., and Mankoff D.A., Quantitative Fluoroestradiol Positron Emission Tomography Imaging Predicts Response to Endocrine Treatment in Breast Cancer. Journal Of Clinical Oncology 24: 2793-2799 (2006)
  70. Green, G.L., Gilna, P., Waterfield, M., Baker, A., Hort, Y., and Shine, J. (1986) Science (Washington D.C.) 231, 1150.
  71. Stenman, V., Biochim. Biophys. Acta 342, 173 (1974)
  72. Kiesel, W., and Davie E. W. Biochemistry 14, 4928 (1975).
  73. Segrest J., and Jackson, R.L. Methods in Enzymology 28, 54 (1972).
  74. Chang C., Kokontis, J., and Liao, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7211-7215
  75. Tilley W.D., Marcelli, M., Wilson, J. D., and McPhaul, M. J., (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 327-331
  76. Joseph, D. R., Hall, S. H., and French, F. S., (1986) In Binding Proteins of Steroid Hormones (Forest, M. G. and Pugeat, M., eds) Vol. 149, pp 123-135, John Libbey, London
  77. Petra P. H., Que B. G., Namkung P. C., Ross J. B. A., Charbonneau H., Walsh K. A., Griffin P. R., Shabanowitz J., and Hunt D. F., Affinity labelling, molecular cloning, and comparative amino acid sequence analyses of Sex steroid Binding Protein of plasma. Ann. N. Y. Acad. Sci. 538 (1988) 10-24
  78. Namkung P. C., and Petra P. H. in Binding Proteins of Steroid Hormones (edited by M. G. Forest and M. Pugeat), Colloque/INSERM. John Libbey, London, Vol. 149 (1986) p. 619.
  79. Petra P.H., Stanczyk F.Z., Senear D.F., Namkung P.C., Novy M.J., Ross J.B.A., Turner E., and Brown J.A.: Current status of the molecular structure and function of the plasma sex steroid-binding protein (SBP). J. Ster. Bioch. 19, 699-706, 1983.
  80. Grishkovskaya, I., Avvakumov, G. V., Sklenar, G., Dales, D., Hammond, G. L., and Muller, Y. A., (2000) EMBO J. 19, 504-512
  81. Grishkovskaya, I., Avvakumov, G. V., Hammond, G. L., and Muller, Y. A., J. Mol. Biol. (2002) 318, 621-626
  82. Grishkovskaya, I., Avvakumov, G. V., Hammond, G. L., Catalano, M. G., and Muller, Y. A., (2002) J. Biol. Chem. 277, 32086-32093
  83. Avvakumov, G. V., Grishkovskaya, I., Muller, Y. A., and Hammond, G. L., (2002) J. Biol. Chem. 277, 45219-45225
  84. Avvakumov, G. V., Grishkovskaya, I., Muller, Y. A., and Hammond, G. L., (2001) J. Biol. Chem. 276, 34453-34457
  85. Thomas S. Kuhn, The Structure of Scientific Revolutions, The University of Chicago Press, fourth edition 2012.
  86. Zakharov, M. N., Bhasin, S., Travison, T. G., Xue, R., Ulloor, J., Vasan, R. S., Carter, E., Wu, F., Jasuja, R., Molecular and Cellular Endocrinology (2015) 399 190-200
  87. Jasuja, R., Spencer, D., Jayarav, A., Peng, L., Krishna, M., Lawney, B., Patel, P., Jayaram, B., Thayer, K. M., Beveridge, D. L., and Bahsin, S., (2021) iScience 24, 102414-102429

Reviews

Neurath H., Bradshaw R.A., Ericsson L.H., Babin D.R., Petra P.H., Walsh K.A.: Current status of the chemical structure of bovine pancreatic carboxypeptidase A. Brookhaven Symposium in Biology 21, 1-23, 1968.

Neurath H., Bradshaw R.A., Petra P.H., and Walsh K.A.: Bovine carboxypeptidase A. Activation, chemical structure, and molecular heterogeneity. Proc. Royal Soc. (London) B-257, 159-176, 1970.

Petra P.H.: Bovine procarboxypeptidase and carboxypeptidase A. Methods in Enzymol. 19, 460-503, 1970.

Petra P.H.: The serum sex steroid-binding protein. Purification, characterization, and immunological properties of the human and rabbit proteins. J. Ster. Bioch. 11, 245-252, 1979.

Petra P.H., Namkung P.C., Titani K., and Walsh K.A. in: Binding Proteins of Steroid Hormones (Forest, M.G., and Pugeat, M., eds) Characterization of the plasma sex steroid-binding protein, Colloque/INSERM, Vol. 149, pp. 15-30, 1986, John Libbey, London/Paris.

Petra, P.H. The Plasma Sex Steroid Binding Protein. A critical review of recent developments on the structure, molecular biology, and function. J. Steroid. Biochem. Mol. Biol. 40: 735-753 (1991).

Personal Data

I was born in Paris in 1937 and came to the United States in 1950 at the age of 13. My father, Yvon Petra, was a highly ranked tennis player in France before the war and reached a ranking of No. 4 in the world after winning Wimbledon in 1946 in singles, and the French Open in doubles in 1937 and 1946. In 2016 he was inducted posthumously into the International Tennis Hall of Fame in Newport, RI, which I attended 1. He turned professional in 1947 and brought the family to the United Sates in 1950. I had learned tennis under his guidance in Belgium in 1947. I won the City of Chicago Tennis Championship in the Boys division (15 and under) in 1952, and reached the quarter finals of the United States Nationals Boys Tennis Championship in 1952 held in Kalamazoo, MI. In 1954, I reached the Finals of the Illinois State juniors Tennis Championship, and in 1959 won the New England Public Parks Open Tennis Championship in Hartford CT and reached the Finals of the Rhode Island Open State Tennis Championship in Providence. I went to Highschool in Palm Beach, Florida, and graduated in 1956. I was offered a tennis athletic scholarship at Tulane University in 1956 and graduated with a BS in chemistry in 1960. I attended Tulane University graduate school in 1960 and obtained an MS and PhD in biochemistry in 1966. Dolores and I became American citizens in 1962 and we were married that same year. In 1966 I was offered a postdoctoral position by Professor Hans Neurath, Chairman of the Biochemistry Department at the University of Washington. We moved to Seattle, and I continued playing tennis and won the Seattle City/Seafair Open Championship in 1968. I was offered a faculty position in the Department of Biochemistry and the Department of Ob/Gyn at the University of Washington in 1971. I stopped tennis competition but returned to it in 2001 at the time of my partial retirement. I fully retired in 2006 but remained active as Professor emeritus of biochemistry to the present.

1  https://www.newportri.com/story/sports/2016/07/21/after-hall-this-time/12778704007/

Selected Pictures

My Dad, my sister and I. Deauville, France, 1946.

My Dad, my sister and I. Deauville, France, 1946.

 

The College years: 1956-1960.
NCAA team champions, 1959. Tulane University, New Orleans.

 

The postdoctoral years: 1966-1970
Ralph and Penny Bradshaw, Dolores, and I. Bateaux Mouches, Paris 1969

 

The faculty years: 1970-2006
UW president McCormick visits my BIOC 406 class.

 

Organizing committee of the
First International Symposium
on steroid Binding Proteins.
Lyon France, 1986.

 

The sabbatical year: 1985
With Etienne Baulieu

 

The Retirement Years: 2006 – present
No more grant writing!